Purification and characterization of an aromatic L-amino acid decarboxylase from Penicillium
raistrickii isolate H10BA2, the transformation of this fungus, and investigation of taxa-4(5),
11(12)-diene synthase
by Bret Reger Niedens
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Plant Pathology
Montana State University
© Copyright by Bret Reger Niedens (1997)
Abstract:
An aromatic L-amino acid decarboxylase from Penicillium raistrickii isolate H10BA2, isolated from
Taxus brevifolia, was purified and characterized. This is the first time this enzyme class has been
described in a fungus. The enzyme was purified to homogeneity by chromatography on
phenyl-sepharose and carboxymethyl-sepharose media. Purity was demonstrated by the presence of
one protein band in a silver stained SDS-PAGE gel. Optimum pH for catalysis by this enzyme is pH
5.0 to 5.6. The enzyme has a broad substrate specificity, utilizing tryptophan (Km= 49 μM), tyrosine
(Km= 1064 úM), and phenylalanine (Km= 99 μM), as well as o-fluorophenylalanine,
p-fluorophenylalanine, and phenylalanine methyl ester. Enzyme activity was not detected with either
5-hydroxytryptophan or histidine as substrate. The protein is a monomer of ' 125,000 (± 3,000) Da, as
determined by reducing SDS-PAGE analysis. The protein has a pl between 6.2 - 6.4, as determined by
enzyme activity being present in this pH range within an isoelectric focusing gel. This fungal aromatic
amino acid decarboxylase is not inhibited by the suicide inhibitors L-alpha-fluoromethyltyrosine and
L-alpha-fluoromethyl(3,4-dihydroxyphenyl)alanine. This enzyme is compared to previously described
aromatic amino acid decarboxylases, and its possible function in the fungus is discussed. P. raistrickii
ATCC#46878, isolated from Eucalyptus sp. leaves, was examined for enzyme activity using the same
techniques as described for P. raistrickii isolate H10BA2. Enzyme activity was not detectable in P.
raistrickiii ATCC#46878.
A transformation protocol was developed for P. raistrickii isolate H10BA2. This fungus was
transformed with the plasmid pAN71, which contains a selectable hygromycin B resistance gene.
Putative transformants were isolated on hygromycin B containing media. Conidia, from colonies of
putative transformants grown on nonselective media, also gave rise to colonies on selective media. This
indicates that stable transformants of P. raistrickii isolate H10BA2 can be developed with the
transformation protocol as described. These transformation experiments were performed to test the
possibility of future determination of aromatic amino acid decarboxylase function in the fungus.
Attempts to identify taxa-4(5), 11 (12)-diene synthase from P. raistrickii isolate H10BA2 with
techniques used for identification this enzyme from Taxus brevifolia are described. PURIFICATION AND CHARACTERIZATION OF AN AROMATIC L-AMINO ACID
DECARBOXYLASE FROM Penicillium raistrickii ISOLATE H10BA2, THE
TRANSFORMATION OF THIS FUNGUS, AND
INVESTIGATION OF TAXA-4(5), 11(12)-DIENE SYNTHASE
By
Bret Reger Niedens
A thesis submitted in partial fulfillment of the requirements for the degree
of
Doctor of Philosophy
in
Plant Pathology
MONTANA STATE UNIVERSITY-BOZEMAN
Bozeman, Montana
September 1997
|s|
APPROVAL
of a thesis submitted by
Bret Reger Niedens
This thesis has been read by each member of the thesis committee and has
been found to be satisfactory regarding content, English usage, format, citations,
bibliographic style, and consistency, and is ready for submission to the College of
Graduate Studies.
- 3 ln M .
John Sherwood
Datfe
Approved for the department of Plant Pathology
Don Mathre
S igna ture )
Date
Approved for the College of Graduate Studies
Joseph J. Fedock
^nature)
Date
1
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a doctoral
degree at Montana State University-Bozeman, I agree that the Library shall make
it available to borrowers under the rules of the Library. I further agree that
copying of this thesis is allowable only for scholarly purposes, consistent with
"fair use” as prescribed in the U.S. Copyright Law. Requests for extensive
copying or reproduction of this thesis should be referred to University Microfilms
International, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have
granted “the exclusive right to reproduce and distribute my dissertation in and
from microform along with the non-exclusive right to reproduce and distribute my
abstract in any format in whole or in part.”
Signature
Date
/% /
IV
To my wife
Lori Lee Niedens
ACKNOWLEDGEMENTS
I would like to thank my parents, George and Nancy Niedens, for supporting
me financially for the last several years, and making it possible to pursue my
education.
I would like to thank Montana Tech of The University of Montana for
allowing me to utilize the space and equipment at the school so that I could
perform the research for this thesis.
I would like to thank Drs. Andrea and Don Stierle of Montana Tech for
allowing me to utilize space, equipment, and supplies within their research group.
They made it possible so that I could perform the research for this thesis. Dr.
Andrea Stierle gave me the opportunity to prove myself as a doctoral candidate.
I would like to thank Steve Parker, at Montana Tech, for the preliminary
research he performed on this enzyme while on sabbatical.
I would like to thank my doctoral thesis committee for the input and advice
they have given me throughout this process.
I would like to thank Dr. Rodney Croteau for allowing me to work in his lab
for a few days to learn the techniques in chapter 4 from Dr. Mehri Hezari and Al
Koepp.
I would like to thank my wife, Lori Lee Niedens, and children, Tera, Rae,
Kent, Amy and Keri, for their support and patience throughout this trying time.
We did it.
TABLE OF CONTENTS
Page
1. INTRODUCTION...................................................................................................1
Background......................................................................................................5
AADC Characteristics................................................................................... 6
Inhibitors of AADC......................................................................................... 11
Decarboxylase Cofactors............................................................................... 12
DNA Sequence Comparisons........................................................................15
Neurotransmitter Formation........................................................................... 16
Melanin Formation...........................................................................................18
Secondary Product Formation in Plants........................................................19
Amines Produced By Fungi........................................................................... 23
Transformation of Penicillium sp................................................................... 23
Applications of AAD C ..............................................................................
30
Taxa-4(5), 11(12)-diene Synthase.................................................................33
2. AROMATIC AMINO ACID DECARBOXYLASE
PURIFICATION AND CHARACTERIZATION...................................................37
Introduction...................................................................................................... 37
Materials and Methods..............................................
37
Fungal Culture.........................................
37
Buffers............................................................. -....................................38
Enzyme Harvest.................................................................................. 39
Enzyme Activity vs. Length of Culture.............................................. 40
Enzyme Purification............................................................................ 40
Hydrophobic Interaction Chromatography................................. 40
Cation Exchange Chromatography.............................................41
Enzyme Assays...................................................................................42
Thin-Layer Chromatography Based Enzyme Assay.................42
Liquid Scintillation Counting Based Enzyme A ssay................. 43
Identification of Enzyme Reaction Product...................................... 44
Gass Chromatography-Mass Spectrometry...............................44
Autoradiography of Thin Layer Chromatogram......................... 44
AADC Utilization of L- or D-Phenylalanine................................
45
Product Formation vs. Length of Enzyme
Assay Incubation................................................................................. 46
Optimization of Temperature............................................................. 46
Optimization of pH...............................................................................47
Optimization of Cofactor Concentration.......................................... 47
Inhibitors of AAD C.......... ....................................................................48
vii
TABLE OF CONTENTS-Continued
2. -Continued
SDS-PoIyacryIamide Gel Electrophoresis........................................48
Isoelectric Focusing to Determine p i................................................ 49
Determination of Km for Substrates..................................................49
Detection of AADC activity in P. raistrickii
ATCC#48678..............
50
50
Results................................................................................
Identification of Enzyme Reaction Product.......................................50
AADC Utilization of L- or D-Phenylalanine.......................................54
Product Formation vs. Length of Enzyme
Assay Incubation................................................................................. 58
Enzyme Activity vs. Length of C ulture...,.......................................... 59
Purification of Aromatic L-Amino Acid Decarboxylase................... 62
Properties of Aromatic L-Amino Acid Decarboxylase.....................64
Effect of Inhibitors on Aromatic L-Amino Acid Decarboxylase.....71
Detection of AADC activity in P. raistrickii ATCC#48678...............71
Discussion.........................................
72
3. TRANSFORMATION OF Penicillium raistrickii................................................. 88
Introduction...................................................................................................... 88
Materials and Methods................................................................................... 89
Harvest of P. raistrickii Conidia.........................................
89
Selection of wild type P. raistrickii mth Hygromycin B.................. .89
Determination of Conditions for the Germination of
P. raistrickii Conidia.............................................................................90
Formation of Protoplasts.................................................................... 90
Transformation Protocol.....................................................................90
Results.......... .................................................................................
92
Harvest of P. raistrickii Conidia...................................................... 92
Selection of wild type P. raistrickii with Hygromycin B....................93
Determination of Conditions for the Germination of
P. raistrickii Conidia.............................................................................93
Formation of Protoplasts................................................
94
Transformation Protocol..................................................................... 94
Discussion........................................................................................................95
viii
TABLE OF CONTENTS-Continued
4. INVESTIGATIONS OF TAXADIENE SYNTHASE ACTIVITY IN
Penicillium raistrickii ISOLATE H10BA2................................................................. 99
Introduction.......................................................................................................99
Materials and Methods.................................................................................... 99
Fungal Culture...................................................................................... 99
Chemicals............................................................................................100
Buffers...... ......................................................
100
Harvest of Protein...............................................................................101
Anion Exchange Chromatography................................................... 102
Enzyme Assay............. .....................................................................102
First Attempt to Identify Enzyme Product........................................103
Second Attempt to Identify Enzyme Product.................................. 104
Effect of pH on Enzyme Activity......... ..............................................106
Identification of Enzyme Product......................
106
Results...........................................................
107
First Attempt to Identify Enzyme Product........................................107
Second Attempt to Identify Enzyme Product............................... 108
Effect of pH on Enzyme Activity....................................................... 114
Identification of Enzyme Product......................................................115
Discussion..........................................................
128
5. DISCUSSION....................................................................................................... 132
REFERENCES CITED.............................................................................................142
ix
LIST OF TABLES
Table
Page
1. Peaks of high specific enzyme activity per gram
mycelial wet weight from mycelia grown in three
different carbon sources...................................................................... 59
2. Purification of aromatic amino acid decarboxylase from
one purification procedure................................................
62
3. Decrease in specific activity in purified enzyme by
freeze/thaw cycle between two subsequent
experiments of enzyme activity.............................................................. 64
4. Comparison of aromatic amino acid decarboxylase
from different sources............................................................................. 70
5. Comparison of effects of a-fluoromethyl analogues
on aromatic amino acid decarboxylase activity
from different sources............................................................................. 71
6. Selection of Penicillium raistrickii conidia on mycological
agar plates containing hygromycin........................................................ 93
7. Determination of germination of R raistrickii conidia
in mycological media with different
concentrations of glycerol added........................................................... 94
8. Formation of protoplasts from germinating conidia
of P. raistrickii in different osmotica.................................................... 94
LIST OF FIGURES
Figure
Page
1. Compounds produced by Penicillium raistrickii isolate
H 1 0 B A 2 ........................................................................................................ 4
2. The reaction performed by aromatic amino acid
decarboxylase and some of the substrates
utilized by this enzyme.................................................................................7
3. Bonds that are cleaved in substrates of enzymes with
pyridoxal-5-phosphate as a cofactor..........................................................8
4. Reaction mechanism of pyridoxal-5-phosphate and
an amino acid in a decarboxylation reaction........................................... 9
5. Examples of aromatic amino acid decarboxylase inhibitors........................12
6. Proposed reaction mechanism of the suicide inhibitor
a-fluoromethyltyrosine with pyridoxal-5-phosphate
cofactor and enzyme...................................................................................13
7. Postulated role of the pyruvyl moiety of histidine decarboxylase............... 14
8. Neurotransmitters that are produced by the
action of aromatic amino acid decarboxylase......................................... 17
9. The reaction performed by tyrosinase............................................................19
10. Secondary products formed by plants with M D C
participation in the biosynthetic pathway................................................. 20
11.
Pathways of auxin synthesis from tryptophan in plants.......................... ...22
12. Amines that are produced by fungi which may be
synthesized via aromatic amino acid decarboxylase activity..............24
13. An example of how homologous recombination
can result in the disruption and inactivation of
a functional gene within a fungus............................................................. 30
xi
LIST OF FIGURES-Continued
14. The toxic 4-methyl tryptophan analogue of tryptophan
is converted to the non-toxic 4-methyl tryptamine by
Catharanthus roseus tryptophan decarboxylase.................................... 32
15. Biosynthesis of taxol from geranylgeranylpyrophosphate
and taxa-4(5), 11(12)-diene....................................................................... 34
16. Comparison of phosphatase and taxa-4(5),
11 (12)-diene synthase reactions...............................................................35
17. Thin-layer chromatography analysis of enzyme products.......................... 52
18. Comparison of a thin-layer chromatogram and the
corresponding autoradiogram of the reaction products
of an aromatic amino acid decarboxylase assay................................... 53
19. Mass spectrum of derivatized tryptamine extracted from an
aromatic amino acid decarboxylase assay..:..........................................55
20. Mass spectrum of a derivatized tryptamine standard.................................. 56
21. Thin-layer chromatography analysis of phenethylamine
production in enzyme assays utilizing L- or
D-phenylalanine as substrate.................................................
57
22. Concentration of tryptamine produced by
phenyl-sepharose active fraction at different
times of incubation......................................................................................58
23. Concentration of tryptamine produced by purified enzyme at different
times of incubation......................................................................................59
24. Aromatic amino acid decarboxylase activity from mycelia of
P. raistrickii grown in three different media during a 20 day
culture.....................................................................
60
25. Mycelial "wet" weight compared to aromatic amino acid
decarboxylase activity........................
61
26. Non-reducing SDS-PAGE (6% polyacrylamide) analysis of enzyme
purification steps.........................
63
xii
LIST OF FIGURES-Continued
27. Effect of temperature on aromatic amino acid
decarboxylase activity.............................................................................. 65
28. Effect of pH on AADC activity measured in two different buffers.............. 65
29. Effect of pyridoxal phosphate concentration on the
activity of extensively dialysed aromatic amino
acid decarboxylase.....................................................................................66
30. Double reciprocal plot of hydrophobic interaction chromatography
active fraction with tryptophan as substrate........................................... 66
31. Double reciprocal plot of hydrophobic interaction chromatography
active fraction with phenylalanine as substrate......................................67
32. Double reciprocal plot of hydrophobic interaction chromatography
active fraction with tyrosine as substrate................................................67
33. Aromatic amino acid decarboxylase activity in gel slices from the
isoelectric focusing electrophoresis experiment for the
determination of the isoelectric point............................................................. 68
34. SDS-PAGE (6% polyacrylamide slab gel) analysis of aromatic
amino acid decarboxylase......................................................................... 69
35. Reaction performed by phenylalanine-2, 3-aminomutase.......................... 72
36. Demonstration of the proposed difference between
L-phenylalanine and D-phenylalanine, and the spacial
relationship between the respective amine groups to the
aldehyde group of pyridoxal-5-phosphate in the
enzyme active site....................
79
37. Three dimensional comparison of a-fluoromethyltyrosine and tyrosine...86
38. Results from liquid scintillation counting analysis of enzyme assays
of anion exchange fractions from the first
attempt to identify enzyme product........................................................108
xiii
LIST OF FIGURES-Continued
39. DPM from the hexane rinse of the silica columns from
enzyme assays of selected fractions from an
anion exchange column described in second
attempt to identify enzyme product.......................................................109
40. DPM from the ethylacetate rinse of the silica
columns from enzyme assays of selected fractions
from an anion exchange column described in second
attempt to identify enzyme product.......................................................110
41. GC-MS analysis of products from the second
attempt to identify enzyme product......................................................111
42. Fragmentation pattern of a peak between 16.901 and
16.921 min from the analysis in figure 41.............. ..............................112
43. Structure of verticillol and it’s mass spectral
fragmentation pattern taken from the work of
Bengt Karlsson et. al. (89).................................................... ...... ...... 113
44. Effect of pH on enzyme activity................................................ , ................ 114
45. The total ion count analysis of a taxa-4(5),
11(20)-diene [20-13C] standard (peak 1531)..........................................116
46. The fragmentation pattern of a shoulder of the
taxa- 4(5), 11(20)-diene [20-13C] standard,
shown in figure 44..................................................................................... 117
47. The total ion count analysis of a geranylgeraniol
standard (peak 1909)................................................................................ 118
48. The fragmentation pattern of the geranylgeraniol
standard, shown in figure 46.................................................................. 120
49. GC-MS analysis of the ethyl acetate rinse, of a silica column
from an AE-150 enzyme assay performed at pH 5.5........................ 121
xiv
LIST OF FIGURES-Continued
50. The fragmentation pattern of the 1906 peak,
shown in figure 4 8 ....................................................................................122
51. GC-MS analysis of the ethyl acetate rinse of a silica column
from a boiled AE-150 enzyme assay control performed
at pH 5.5.................................................................................................... 123
52. GC-MS analysis of the ethyl acetate rinse of a silica
column from an AE-150 enzyme assay performed at pH 8.5............125
53. The fragmentation pattern of the 1906 peak, shown in figure 51.......... 126
54. GC-MS analysis of the ethyl acetate rinse of a silica
. column from a boiled AE-150 enzyme assay
control performed at pH 8.5.....................................................................127
XV
ABSTRACT
An aromatic L-amino acid decarboxylase from Penicillium raistrickii isolate
H10BA2, isolated from Taxus brevifolia, was purified and characterized. This is
the first time this enzyme class has been described in a fungus. The enzyme
was purified to homogeneity by chromatography on phenyl-sepharose and
carboxymethyl-sepharose media. Purity was demonstrated by the presence of
one protein band in a silver stained SDS-PAGE gel. Optimum pH for catalysis by
this enzyme is pH 5.0 to 5.6. The enzyme has a broad substrate specificity,
utilizing tryptophan (Km= 49 pM), tyrosine (Km= 1064 jaM), and phenylalanine
(Km= 99 pM), as well as o-fluorophenylalanine, p-fluorophenylalanine, and
phenylalanine methyl ester. Enzyme activity was not detected with either 5hydroxytryptophan or histidine as substrate. The protein is a monomer of '
125,000 (± 3,000) Da, as determined by reducing SDS-PAGE analysis. The
protein has a pi between 6.2 - 6.4, as determined by enzyme activity being
present in this pH range within an isoelectric focusing gel. This fungal aromatic
amino acid decarboxylase is not inhibited by the suicide inhibitors L-alphafluoromethyltyrosine and L-alpha-fluoromethyl(3,4-dihydroxyphenyl)alanine. This
enzyme is compared to previously described aromatic amino acid
decarboxylases, and its possible function in the fungus is discussed. P. raistrickii
ATCC#46878, isolated from Eucalyptus sp. leaves, was examined for enzyme
activity using the same techniques as described for P. raistrickii isolate H10BA2.
Enzyme activity was not detectable in P. raistrickiii ATCC#46878.
A transformation protocol was developed for P. raistrickii isolate H10BA2.
This fungus was transformed with the plasmid pAN71, which contains a
selectable hygromycin B resistance gene. Putative transformants were isolated
on hygromycin B containing media. Conidia, from colonies of putative
transformants grown on nonselective media, also gave rise to colonies on
selective media. This indicates that stable transformants of P. raistrickii isolate
H10BA2 can be developed with the transformation protocol as described. These
transformation experiments were performed to test the possibility of future
determination of aromatic amino acid decarboxylase function in the fungus.
Attempts to identify taxa-4(5), 11 (12)-diene synthase from P. raistrickii
isolate H10BA2 with techniques used for identification this enzyme from Taxus
brevifolia are described.
1
CHAPTER 1
INTRODUCTION
Aromatic L-amino acid decarboxylases (AADCs,1E.C.4.1.1.28) catalyze the
decarboxylation of various aromatic L-amino acids, yielding the respective
amines and carbon dioxide. These enzymes have been described from several
organisms, with each enzyme having unique characteristics. These AADCs vary
in the range of and affinity for aromatic amino acids that can be used as
substrates. Sources from which this enzyme have been described include
mamrpals (1, 2, 3, 4, 5), plants (6, 7, 8, 9, 10, 11, 12), fish (13), insect (14), and
bacteria (15, 16, 17). This class of enzyme has not been described from a
fungus. The purpose o f this paper is to report the purification and
characterization of an AADC from the fungus Penicillium raistrickii isolate
H10BA2 (18), which was isolated from Taxus brevifolia, and compare it to
enzymes from different sources. P. raistrickii ATCC # 48678, which was isolated
1 The abbreviations used are: A A D C 1 aromatic L-amiho acid decarboxylase; D O P A 1 3,4dihydroxyphenylalanine; Pxy-P, pyridoxal 5-phosphate; G C -M S 1 gas chromatography mass
spectrometry; S D S 1 sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; 5-H TP,
5-hydroxytryptophan.
2
from Eucalyptus, was also examined for AADC activity. This was done to
determine if AADC activity was ubiquitous within the species P. raistrickii
To determine the function of AADC in P. raistrickii, it will be necessary to
remove the enzyme activity from the fungus. This can be accomplished by
transformation of P. raistrickii isolate H10BA2 with foreign DNA. The foreign
DNA would contain a disrupted version of the AADC gene that would result in
disruption of the AADC gene within the P. raistrickii genome via homologous
recombination. Preliminary experiments to transform P. raistrickii with plasmid
DNA will also be described.
AADCs are involved in anabolic pathways that can lead to a variety of
compounds. It is involved in the biosynthesis of indoleamines (serotonin and
melatonin), catecholamines (dopamine, noradrenaline, and adrenaline),
isoquinoline, and other alkaloids. The function of this enzyme in mammals and
fish is to produce neurotransmitters in neurons, but it's function in
nonmohoamine neuron tissue such as kidney and liver, from which it has also
been localized, is not known (19). AADC in kidney and liver may function in the
metabolism of tyrosine, phenylalanine and tryptophan when the tissue levels of
these amino acids are elevated (20). The function of this enzyme in plants is to
provide precursors for the biosyntheses of various alkaloids with a wide range of
biological activities. In Catharanthus roseus, Papaver somniferum and
Thalictrum rugosum, AADCs are involved in the production of the antineoplastic
compound vincristine (11), the analgesic morphine (9), and the antibacterial
3
berberine (12), respectively. In insects, this enzyme is present in the nervous
system and in the epidermis where it produces dopamine that is required'for
cuticular melanization (21). No biological purpose for the production of aromatic
amines by bacteria has been described, but the production of biogenic amines by
bacteria in food products has been associated with incidents of food poisoning
(22). AADC from Streptococcus faecalis has been used as the basis for assays
for the determination of Pxy-P in biological fluids (15).
The biological function of this enzyme in PeniciIHum raistrickii has not been
determined, but it could be involved in the production of secondary metabolites or
melanization. Aromatic amines, and the alkaloids synthesized from them, have
been isolated from fungi (23). Several of these compounds, such as bufotenine
and psilocybin, have psychoactive biological activity. This Penicillium raistrickii,
isolated from the phloem of Taxus brevifolia, is of interest to our research group
because of the biologically active compounds it produces (18). Compounds that
this fungus produces include the antineoplastic compound paclitaxel and the
tremorgenic penitrems (see figure 1). AADC was purified and characterized
because this is the first time it has been described in a fungus, and this enzyme
may play a critical role in the biosynthesis of natural products. The presence or
absence of AADC activity in P. raistrickii ATCC # 48678, isolated from
Eucalyptus, was also determined, to establish the presence of this enzyme
activity within the species P. raistrickii.
4
Figure 1. Compounds produced by Penicillium raistrickii isolate H10BA2.
6-methoxymellein
\\
OH
pseurotin A
0-CH2CH-C-O-CH 2CHCH 2-0
benzomalvin C
N-benzyoyl-0-[N'-benxoylL-phenylalanylj-L-phenylalaninol
penitrem A R i= C I Rz=OH
penitrem B R i= H Rz=H
5
Experiments involving the transformation of P. raistrickii with plasmid DNA
were also performed. Transformation experiments were performed to develop
the techniques necessary for genetic manipulation of this fungus. Experiments to
identify taxa-4(5) 11(12)-diene Synthase, the first committed step in the
biosynthesis of taxol, from this fungus will also be described.
Background
In this section, I will review in greater detail what is known about AADCs. I
will begin by reviewing the enzyme and its characteristics, DNA sequence
comparisons and what is known about the biological role of the enzyme in
organisms from which it has been isolated. Characteristics of AADCs that will be
discussed are the reaction performed by the enzyme, inhibitors of this reaction
and cofactors used by decarboxylases. Other physical characteristics and a
comparison of AADCs from different organisms will be commented on in the
discussion of the results of this research. The biological role of AADCs in the
biosynthesis of neurotransmitters, melanin and secondary metabolites will be
discussed separately. Amines that have been isolated from fungi and that are
theoretically derived from aromatic amino acid decarboxylase activity in fungi will
be presented. Applications of AADC genes will also be reviewed. A review of
the work on transformation of PeniciIHum sp. and taxa-4(5), 11(12)-diene
synthase characterization will also be presented.
6
AADC Characteristics
Aromatic L-amino acid decarboxylases are an enzyme class that can
catalyze the decarboxylation of both essential and non-essential aromatic amino
acids to produce their corresponding amines, as shown in figure 2. AADCs can
be broad or narrow in the range of amino acids they use as substrates, reflecting
the biological function the enzyme plays in an organism. Though histidine is an
aromatic amino acid, it is not a good substrate for the AADCs that will be
discussed here. Histidine is efficiently decarboxylated by a different enzyme,
histidine decarboxylase. Mammals have a separate decarboxylase for histidine
presumably to facilitate the roles played by histamine in the organism. Histamine
is involved in gastric secretion, peripheral circulation, allergic and similar
hypersensitive reactions, and certain types of rapid growth (24). The necessity
for specific regulation of these types of activities would appear to justify the need
for a histidine decarboxylase separate from other AADC activity. Histidine
decarboxylase is not considered a member of the AADC class of enzymes for
these reasons. The range of amino acids utilized by the different AADCs will be
discussed in greater detail during the discussion of the enzymes biological role in
the organism.
To perform the decarboxylation reaction, AADCs utilize Pxy-P as a cofactor.
Pxy-P functions as a cofactor in a variety of reactions involving a-amino acids.
Reactions in which it participates include transaminations, a decarboxylations,
7
Figure 2. The reaction performed by aromatic amino acid decarboxylase and
some of the substrates utilized by this enzyme.
R .-X V NH2
COOH
R=
Phenylalanine
Tryptophan
Tyrosine
Histidine
HO
3,4-dihydroxyphenylalanine
5-hydroxytryptophan
F
<
>
o-fluorophenylalanine
p-fluorophenylalanine
O
CH3
phenylalanine methyl ester
8
aldolizations, and the p decarboxylation of aspartic acid (25). The bonds that can
be cleaved by participation of Pxy-P in an enzyme are demonstrated in figure 3.
Figure 3. Bonds that are cleaved in substrates of enzymes with
pyridoxal-5-phosphate as a cofactor. ( - — ) cleaved bonds. (24)
(SR)
SRORH
:
I
i
H
NH
O
Il
The reaction sequence for decarboxylation of an amino acid is presented in
figure 4. The function of Pxy-P is to stabilize the formation of a carbanion at the
a and p carbons of a-amino acids. This is accomplished by the electrophilic
nitrogen of the pyridine ring which helps form the stable resonance structure
shown in figure 4.
The roles of some amino acids within rat liver AADC in the catalysis of the
reaction have been investigated (26). Amino acids that might participate in the
reaction were identified after aligning the amino acid sequences of 13 amino acid
decarboxylases. Amino acids that were invariant or conservatively substituted in
the sequences were changed by site directed mutagenesis and expressed in
Escherichia coli. The enzyme activity of the resulting proteins was then
compared to the native enzyme. Amino acids that were investigated in rat liver
9
Figure 4. Reaction mechanism of pyridoxal-5-phosphate and an amino acid in a
decarboxylation reaction. R= aromatic functional group shown in figure 2.
H H O
R— c — c — c — O"
I
H H O
I I Il
I
H HN+H
H
R— C— C— C— O"
CH
Pyridine Nitrogen that
Schiffs base or Aldimine
H HN+ H
H
10
AADC include lysine (Lys) at amino acid residue 303, arginine (Arg) 355 and
aspartic acid (Asp) 271 (26).
In previous studies it had been shown that Lys 303 binds the coenzyme
Pxy-P (27). Pxy-P forms a Schiff base with Lys 303. The Pxy-P then undergoes
a transaldimination reaction with the substrate amino acid to form a Schiff base
(see figure 4). Rat AADC is a dimer and each Lys 303 binds a molecule of PxyP. Other possible functions for Lys 303 include accelerated formation of Pxy-Psubstrate Schiff base and/or displacement of the product from the Pxy-P-product
Schiff base.
In rat AADC1Arg 355 could possibly be involved in binding the
oc-
carboxylate group. When Arg 355 was replaced with Lys, there was no effect on
the enzyme activity, but when it was replaced with alanine (Ala), enzyme activity
disappeared. This suggests that a positive charge is required in rat AADC at
position 355.
As stated previously, Pxy-P acts as a cofactor to stabilize the carbanion
formed at the a-carbon via the electrophilic pyridine nitrogen. Asp 271 may
interact with the pyridine nitrogen of Pxy-P to stabilize the protonated nitrogen
form of Pxy-P (see figure 4). When Asp 271 was replaced by Ala the enzyme
was not active, but when it was replaced by glutamic acid (GIu) there was a
decrease in kCat, but not Km. The decrease in the turnover number, or kcat, means
that the enzyme reaction does not occur as rapidly as before. No change in the
Kmsignifies that the enzyme's affinity for substrate has not changed. A negative
11
charge is therefore required at this position to enhance the catalytic efficiency of
rat AADC (26).
Inhibitors of AADC
Inhibitors of AADCs interact with the Pxy-P associated with the enzyme.
Pxy-P is tightly bound to the enzyme, so reaction with these inhibitors does not
remove the coenzyme from the apoenzyme (28). A few examples of AADC
inhibitors are presented in figure 5. a-Fluoromethyltyrosine acts as a suicide
inhibitor to produce inactive enzyme (29). Suicide inhibitors are intrinsically
unreactive molecules that become reactive after the enzyme has modified them,
and become covalently bound at the enzyme's active site (25). The proposed
mechanism by which a-fluoromethyltyrosine binds covalently to Pxy-P and the
enzyme is shown in figure 6. Inhibitors of AADC activity have medicinal
application for the treatment of hypertension and symptoms of Parkinson’s
disease, which will be described later in the discussion of neurotransmitter
production. Methyldopa is available for the treatment of hypertension under the
prescription name of aldomet, and carbidopa is available for the treatment of
symptoms of Parkinson’s disease under the prescription name of sinemet (30).
12
Figure 5. Examples of aromatic amino acid decarboxylase inhibitors.
HO
/
^
COOH
O -N H 2
a-Aminooxy-p-phenylpropionic acid
HO
ho-
/
COOH
^
H9N
CH9F
a-Fluoromethyltyrosine
Decarboxylase Cofactors
All previously described members of the AADC enzyme class utilize Pxy-P
as a cofactor, but not all decarboxylase enzymes utilize Pxy-P as a cofactor (24).
Some decarboxylase enzymes utilize a pyruvate functional group to stabilize
carbanion formation in the substrate during the reaction (see figure I). These
enzymes have been found only in bacteria. Examples of this type of enzyme
include adenosylmethionine decarboxylase and histidine decarboxylase. In the
histidine decarboxylase of Lactobacillus 30a, the pyruvyl moiety is derived from
the autocatalytic chain cleavage between two serine residues, and one of the
serine residues is converted to the pyruvyl moiety (31).
13
Figure 6. Proposed reaction mechanism of the suicide inhibitor a fluoromethyltyrosine with pyridoxal-5-phosphate cofactor and enzyme (29).
R— c — i
H
R— c — i
CM.
R— C— i
N+
H
R— C— I
CH3
H
14
Figure 7. Postulated role of the pyruvyl moiety of histidine decarboxylase.
Histidine Decarboxylase
with pyruvyl moiety
Histidine
C -O '
HgC— C — C — NH— Enzyme
+
Histamine
C -O
HgC— C— C — NH— Enzyme
HgC— C — C— NH— Enzyme
O
15
There have been reports of either one or two molecules of Pxy-P being
present in the holoenzyme. The natural form of pig kidney AADC binds one
molecule of Pxy-P per apoenzyme, as opposed to the recombinant form
expressed in Escherichia coli that binds two molecules of Pxy-P per enzyme
(32). Rat AADC is a dimer and each Lys 303 binds a molecule of Pxy-P (26).
DNA Sequence Comparisons
Several AADC genes have been cloned and sequenced, and some
sequence comparisons at the DNA and amino acid levels have been reported. In
1989, the deduced amino acid sequence of an AADC cDNA sequence from the
plant Catharanthus roseus was shown to have an identity of 39% to Drosophila
melanogaster (fruit fly) AADC134% to Drosophila melanogaster a-methyldopa
hypersensitive gene, 19% to feline glutamate decarboxylase and 12% to mouse
ornithine decarboxylase (33). The similarities extended throughout the protein
sequence. Also in 1989, the sequence for a rat AADC cDNA was reported (34).
It was found that this enzyme contained two regions of amino acid sequence that
were similar to each other in 14 of 61 positions. These regions were then
compared to similar regions in other enzymes. It was found that these regions
showed an identity of 27.6 % to rat tyrosine hydroxylase, 24.1% to human
dopamine (5-hydroxylase, and 24.1% to bovine phenylethanolamine Nmethyltransferase (34). Later in 1989, the AADC cDNA sequence for humans
I
16
was reported (35). The deduced amino acid sequence showed a 59% identity
and 77% similarity to Drosophila melanogaster AADC after optimal alignment
was made (35). In 1994, a gene family of AADCs in opium poppy was reported
(36). The deduced amino acid sequences of gTYDCI and cTYDC2 showed an
identity of 64% and 62% to the AADC of parsley, 52% and 51 % to AADC of the
Catharanthus roseus, and 38% and 37% to AADC of Drosophila melanogaster,
respectively (36). In 1995, the AADC from Manduca sexta (tobacco hornworm)
was reported and showed a 64% identity at the DNA level and a 72% identity at
the amino acid level to the AADC of Drosophila melanogaster (37). These
comparisons demonstrate that there are similarities in sequence between
kingdoms for this enzyme. A specific sequence common to all is the Pxy-P
binding region Pro-His-Lys contained in each monomer of the homodimer
holoenzyme.
Neurotransmitter Formation
Certain AADCs in mammals and fish catalyze the formation of serotonin
and dopamine, which are important neurotransmitters in the central nervous
system (see figure 8) (19). Dopamine can then be converted to epinephrine and
norepinephrine. The occurrence of AADCs in mammals and fish is associated
with this activity, but AADCs can also be isolated from liver and kidney, tissues
that do not contain monoamine neurons. The function of AADCs in these tissues
17
is not known, but may be involved in the metabolism of tyrosine, phenylalanine,
and tryptophan when the tissue levels of these amino acids is elevated (20).
Mammalian and fish AADCs demonstrate a higher affinity for DORA and 5hydroxytryptophan than the other amino acids (see table A). This reflects the
function of this enzyme in the biosynthesis of neurotransmitters that are derived
from these amino acids.
Figure 8. Neurotransmitters that are produced by the action of aromatic amino
acid decarboxylase.
Serotonin
Epinephrine
Dopamine
Norepinephrine
Two medical disorders associated with this AADC are Parkinson’s disease
and hypertension. Parkinson’s disease symptoms are associated with depletion
of dopamine in the corpus striatum region of the brain. To treat these symptoms,
DOPA (see figure 2) is administered with carbidopa (see figure 5). Carbidopa is
an inhibitor of peripheral AADCs and does not cross the blood brain barrier,
thereby allowing DOPA, which does pass through the blood brain barrier, to enter
18
the central nervous system and be decarboxylated into dopamine. Hypertension
is treated with the inhibitor methyldopa. The specific method by which
methyldopa lowers arterial pressure is not known, but it is theorized that this is
accomplished by stimulation of central inhibitory alpha-adrenergic receptors,
false neurotransmission, and/or reduction of plasma renin activity (30).
Melanin Formation
Melanins are pigments consisting of highly irregular polymers. AADCs can
function in the formation of melanin or melanization in insects. This activity is
crucial in the maturation process of insect cuticular melanization (38).
Melanization is also part of the immune response and wound healing in insects.
The amino acid utilized in this process is DORA, which is decarboxylated to form
dopamine, which is further metabolized to form melanin. This is reflected in the
specificity of insect AADCs for DORA as substrate. DORA is the only amino acid
utilized by this enzyme (14). This is in contrast to fungal melanin biosynthesis in
which tyrosinase oxidizes DORA to form dopaquinone (see figure 9) (39).
Dopaquinone either feeds directly into melanin formation or is further
metabolized and then incorporated into fungal melanin. Other precursors for
melanin formation in fungi can include y-glutaminyl-4-hydroxybenzene, catechol
and 1,8-dihydroxynaphthalene. This does not mean that an AADC cannot be
19
involved in melanin formation in fungi. Formation of melanin in fungi with the
participation of an AADC has not been described.
Figure 9. The reaction performed by tyrosinase.
HO
H .-'
NH9
Tyrosinase
COOH
3,4-dihydroxyphenylalanine
Secondary Product Formation in Plants
AADCs also participate in the production of secondary metabolites in
plants. Figure 10 lists some of the compounds that are synthesized from amines
derived from the decarboxylase reaction. Serotonin is produced by Juglans regia
and Cytisus scoparius^O, 7). Strictosidine and salidroside are precursors to the
biologically active compounds vincristine and verbascoside, respectively. As can
be seen from figure 10, amines resulting from AADCs can be incorporated into a
variety of compounds with biological activity.
Isoquinoline alkaloids, such as morphine, berberine and sanguinarine, are
derived from tyrosine (figure 10). The AADCs from Eschscholtzia californica and
Thalictrum rugosum are associated with production of the isoquinoline alkaloids
sanguinarine and berberine, respectively. The AADC from Eschscholtzia
californica uses only tyrosine as substrate while the AADC from Thalictrum
20
Figure 10. Secondary products formed by plants with AADC participation in the
biosynthetic pathway.
O
Harman from P assiflora edulis
-\
Morphine from
Papaver som niferum
_____
v N" c h 3
HO
^
%
N1N-Dimethyltryptamine from
Phalaris tub eras a
Hordenine from
Hordeum sativum
Salidroside from
Syringa vulgaris
Stictosidine from Catharanthus roseus
Glc = glucose
H,CO
Berbehne from
Thalictm m rugosum
Sanguinarine from
E schscholtzia califom ica
21
rugosum can utilize tyrosine and DOPA (12). Salidroside is synthesized from
tyramine, but does not retain the amine group.
Indoleamines are derived from tryptophan and include harman, N1Ndimethyltryptamine, and strictosidine (figure 10). Strictosidine is produced by
Catharanthus roseus as part of the biosynthetic pathway to vincristine. The
AADC from Catharanthus roseus only utilizes tryptophan and 5hydroxytryptophan as substrates. This reflects the function of this enzyme in the
biosynthesis of indoleamines.
AADCs have been identified in Arabidopsis thaliana and Petroselinum
crispum as being pathogen responsive genes (40, 41). In Petroselinum crispum,
cDNAs coding for pathogenesis-related and elicitor-activated genes were
identified in cell cultures that were exposed to fungal elicitors from the soybean
pathogen Phytophthora megasperma f. sp. glycinea (42). Some of these
pathogenesis-related genes were shown to code for a tyrosine decarboxylase
(41). This activity as a pathogenesis-related gene is thought to be associated
with the production of isoquinoline alkaloids.
lndole-3-acetic acid, one of the major phytohormones, is synthesized from
tryptophan (see figure 11)(43). There are at least two pathways for the synthesis
of indole-3-acetic acid. Enzyme activities for both of the pathways shown in
figure 11 have been detected in plants (44, 45).
22
Figure 11. Pathways of auxin synthesis from tryptophan in plants.
Tryptophan
COOH
___
NH2
NH3
c
V
Tryptophan
Transaminase
1/2 0 2
Tryptophan
Decarboxylase
lndole-3-pyruvate
Tryptamine
___
NH2
NH3
Amine
Oxidase
CO2^ y
1/20 2
A
Indolepyruvate
Decarboxylase
lndole-3-acetaldehyde
-------- ^ C H
II
0
1/2 O2
Indoleacetaldehyde
V Dehydrogenase
lndole-3-acetic acid
X X _____ 'COOH
COOH
23
Amines Produced by Fungi
There are several indoleamines produced by fungi, as shown in figure 12
(23). The amines that are unique to fungi are psilocin and psilocybin that are
produced by Psilocybe spp. The other amines in figure 12 are produced by
plants as well. These compounds are of interest because of the biological
activities associated with them. Bufotenine, psilocin, psilocybin,
dimethyltryptamine are listed as hallucinogens and are controlled substances.
Tryptamines are associated with a condition called Phalaris staggers in Australia
and bovine pulmonary emphysema in Canada when cattle feed on Phalaris
arundinacea. 5-Hydroxytryptamine is considered to be one of the active
principles in the stinging hairs of some plants. Another indoleamine produced by
a fungus, Rhizopus suinus, is indole-3-acetic acid (figure 10) (46). All of these
indoleamines are potentially synthesized from the activity of AADC.
Transformation of Pehicillium so.
Penicillium species are of interest industrially because of the natural
products (penicillins) and enzymes (amylases and galactosidases) which they
produce. To isolate and study the expression of the genes involved in the
production of these metabolites it has been necessary to develop transformation
protocols for Penicillium. Two ways described for the introduction of DNA into
24
Figure 12. Amines that are produced by fungi which may be synthesized via
aromatic amino acid decarboxylase activity.
Dimethyltryptamine
Tryptamine
_____
NH2
N -C H 3
CH3
H
H
5-Hydroxytryptam i ne
Bufotenine
NH2
X
N -C H 3
CH3
j- V
H
H
Methylserotonin
Methoxy N-dimethyltryptami ne
NH
CH3
N-CH3
CH3
H
H
Psilocybin
Psilocin
0
HO-P-OH
1
OH
O
N -CH3
CH3
H
N -CH3
CH3
H
25
Penicillium are electroporation (47) and polyethylene glycol (PEG) mediated
transformation (48).
Transformation of Penicillium urticae by electroporation involves germinated
conidia treated with (3-glucuronidase. Treatment of germinated conidia with 13glucuronidase weakens the cell walls so that DNA can be introduced into the
cells, but does not result in the formation of protoplasts. The cells can then be
subjected to electroporation as specified (47).
The technique of Yelton et. al. for the PEG mediated transformation of
Aspergillus nidulans is used as the basic method for the transformation of
Penicillium sp. in the majority of papers (49,50, 51, 52, 53, 54, 55). In this
technique, protoplasts are formed from mycelium treated with novozyme 234.
The protoplasts are stabilized in different osmotica at different concentrations.
DNA is first added to the protoplasts, and a buffered solution of PEG and CaCb is
then added to facilitate uptake of the DNA by the cells.
Selection of transformed cells is accomplished by a variety of methods,
including complementation of mutants and selection for drug resistance genes.
Complementation of auxotrophic mutants of P. chrysogenum, the industrial
source of penicillin, has been used to select for transformants. The plasmid
p P ctrp C I, which contains a genomic sequence of the P. chrysogenum trpC
gene, was selected for in transformants of P. chrysogenum mutants that were
auxotrophic at trpC, because of a rearrangement at trpC, on media lacking
tryptophan (49). The plasmid pDJB1 contains a genomic sequence of the
26
Neurospora crassa pyr4 gene that codes for orotidine-5’-monophosphate
decarboxylase, and was selected for in transformants of P. chrysogenum
mutants at pyrG, on minimal media (56). Mutants at pyrG were obtained by
mutagenizing conidia and selecting for resistance to 5-fluoroorotic acid (56). The
plasmid pSTA10 contains a genomic sequence of the A. niger niaD gene that
codes for nitrate reductase and was selected for in transformants of P.
canescens mutants lacking niaD activity. Mutants at niaD were obtained by
mutagenizing conidia and selecting for chlorate resistant mutants (57). In all of
these reports, the investigators had to first isolate fungal: mutants before the
fungus could be used in transformation experiments. Unfortunately, the process
of mutagenesis can result in unfavorable changes in the fungus such as loss of a
desired characteristic and should be avoided if at all possible. For this reason
dominant selectable markers have been developed for the selection of
transformed Penioillium sp. The above reports demonstrate the use of nonpenicillium based DNA sequences in the selectable markers, and show that
Penicillium can express Aspergillus and Neurospora DNA sequences.
Several dominant selectable markers have been developed from
resistance genes to different antibiotics. These genes include the hygromycin
resistance gene hph from Escherichia coli, the phleomycin resistance gene ble of
bacterial origin, and the sulfonamide resistance gene from plasmid R388.
Hygromycin B inhibits protein synthesis by interfering with translocation and
causes misreading (58, 59). The hygromycin resistance gene from E coli
27
inactivates hygromycin by phosphorylation (60). The sulfonamide resistance
gene codes for dihydropteroate synthetase (51). This activity gives resistance to
the drugs sulfanilamide and sulfamethoxazole.
The most widely used resistance gene used for transforming PeniciIHum sp.
is hph, the hygromycin resistance gene. Plasmids based on this resistance gene
are pAN7-1, pCSN44, pDH25MC, and pDH333. The most commonly used
plasmid for the transformation of Penicillium is pAN7-1 (52, 54, 55). This plasmid
contains the promoter sequence for the A. nidulans glyceraldehyde-3-phosphate
dehydrogenase, the E. coli hygromycin resistance gene, and the A. nidulans trpC
terminator sequence (61). This construct allows for the selection of PeniciIHum
transformants on hygromycin B containing media. The plasmid pAN7-1 has
been used to transform P. roquefortii (52), P. paxilli (54), and P. freii (55). The
plasmid pCSN44 also contains the bacterial hygromycin resistance gene and has
been used to transform the phytopathogen P. urticae (47). P. urticae is the
causal agent of blackleg disease of crucifers, and produces the mycotoxins
patulin, patulolide and griseofulvin. The plasmid pDH25MC contains the A.
nidulans trpC promoter, the E coli hph gene, and the A. nidulans trpC terminator
sequence. This plasmid has also been used to transform P. urticae (62). The
plasmid pDH333 contains the promoter from A. nidulans pgk, the E. coli hph
gene, and the A. nidulans pgk terminator. The gene pgk codes for, 3phosphoglycerate kinase. This plasmid was used to transform P. citrinum, the
28
producer of compactin. Compactin is an inhibitor of 3-hydroxy-3-methyl-glutaryl
coenzyme A reductase, the rate-limiting step in cholesterol biosynthesis.
The plasmid pUT771 has been used to impart phleomycin resistance. This
plasmid contains a fungal promoter upstream of the phleomycin resistance gene,
phleor, followed by the trpC terminator from A. nidulans (52). This plasmid has
been used to transform P. roquefortii. P. roquefortii is used in the dairy industry .
to make blue cheese.
The plasmid pML2 has been used to impart resistance to sulfonamide
antibiotics. This plasmid contains the P. chrysogenum trpC promoter, the sul\
gene, followed by the P. chrysogenum trpC terminator (51). This plasmid was
used to transform P. chrysogenum.
Transformation efficiencies are reported from 7 to 3,000 transformants per
|j,g DNA for these different fungi with plasmids containing selectable markers (62,
56).
All of the above references report that the plasmids were integrated into the
chromosomes of the fungi. Homologous recombination of the plasmid pPctrpCI
was reported in P. chrysogenum (49).
Homologous recombination is the crossing-over of two DNA sequences
which are similar over a specific region. This is in opposition to non-homologous
recombination, where crossing-over can occur randomly within a genome. The .
specificity of homologous recombination can be used as a tool to insert new DNA
sequences at a specific point within a genome. An example of homologous
recombination to restore a phenotype was reported for P. chrysogenum (49).
29
The fungus used in this experiment contained a DNA rearrangement that
affected the trpC region resulting in a Trp' auxotrophy. The Trp" auxotroph was
then transformed with the plasmid pPctrpCI, which contained a functional trpC
gene. In a few transformants there was a recombination event between the
homologous trpC genes of the fungus and plasmid that resulted'in a Trp+
phenotype (49). The process of homologous recombination can be used to
disrupt a functional gene within a fungus, as shown in figure 13. The example
given in figure 13 describes one method of inactivating a gene within a fungus by
homologous recombination.. The result in figure 13 would produce a fungus that
expresses a resistance gene and non-functional structural gene products. An
example of this use of homologous recombination is reported for A. nidulans
(63).
Autonomously replicating sequences have been used to increase
transformation frequencies. The plasmid pDJB2 is the plasmid pJB1 containing
the ans 1 sequence from A. nidulans. The ans 1 sequence was isolated as an
autonomously replicating sequence (64). The plasmid pDJB2 was reported to
increase the transformation frequency 3-fold and the stability of integrative
transformants. Another report described the use of the plasmid pHELPI that
contains a 5 kb fragment of the AMA1 autonomous replication element from A.
nidulans that resulted in non-integrated forms of the selectable DNA (57).
Cotransformation of linearized pHELPI and pSTAIO resulted in a transformation
frequency of 50,000 transformants per ^g DNA. These transformants were
30
Figure 13. An example of how homologous recombination can result in the
disruption and inactivation of a functional gene within a fungus. The plasmid
contains a version of the structural gene where the asterisks can represent
mutations or deletions, which do not allow a functional gene product to be
produced. A recombination event occurs in the middle of the homologous
sequences, resulting in integration of the plasmid into the fungal genome, and
inactivation of the fungal structural gene.
Recombination event
—
—
cza
□
Genomic DNA
Plasmid DNA
Resistance Gene
Structural Gene
described to contain non-integrated forms of these plasmids and the
transformants lost this DNA when grown under non-selective conditions (57).
Applications of AADC
AADCs have been used for several applications. The first use of AADC
took advantage of the enzyme’s requirement for Pxy-P to determine the
31
concentration of Pxy-P in biological fluids (15). An acetone powder of
Streptococcus faecalis grown in pyridoxine-deficient media was prepared. This
enzyme preparation was then either be purified or used as a source for AADC
activity. The crude enzyme mixture was added to a buffer containing a portion of
a biological sample, such as human plasma, and enzyme activity measured. The
enzyme activity was directly proportional to the concentration of Pxy-P in the
sample. Determination of Pxy-P concentrations in plasma is important since this
cofactor has been implicated in several clinical disorders, including peripheral
neuropathy, convulsions, sideroblastic anemia, and liver disease (65).
AADC has been also used as a selectable marker in plant cells (66). In this
system, a DNA vector was constructed with the cauliflower mosaic virus 35S
promoter expressing the Catharanthus roseus tdc cDNA for tryptophan
decarboxylase. This enzyme converts the toxin 4-methyl tryptophan to the non­
toxic 4-methyl tryptamine, see figure 14. This vector was used to transfect
tobacco cells and allowed for the selection of transgenic cells on 4-methyl
tryptophan containing media. It was also found that expression of this gene in
the progeny of transgenic tobacco plants resulted in the production of tryptamine
and in a reduction of the ability to support whitefly reproduction, when compared
to the control plants (67). Sweetpotato whitefly reproduction was determined by
comparing the emergence of whitefly pupae by counting hatched pupal cases. A
97% decrease in whitefly reproduction was observed relative to controls.
32
Figure 14. The toxic 4-methyl tryptophan analogue of tryptophan is converted to
the non-toxic 4-methyl tryptamine by Catharanthus roseus tryptophan
decarboxylase.
CH3
CH3
NH2
,NH;
COOH
I
H
4-methyl tryptophan
CO2
4-methyl tryptamine
Introduction of the Catharanthus roseus tdc cDNA, under the control of the
cauliflower mosaic virus 35S promoter, into potato did not result in a more
pathogen resistant potato (68). Instead, it was found that introduction of this
gene into potato altered the phenylpropanoid pathway. The phenylpropanoid
pathway leads to the formation of lignin, coumarins and flavonoids. This pathway
can be involved in the plant defense response to wounding and pathogen
infection (69,70). Potatoes expressing this gene were shown to have decreased
levels of phenylpropanoid pathway products that resulted in increased
susceptibility to the pathogen Phytophthora infestans (68).
As described in this background, AADCs serve anabolic functions within the
respective organisms. It is involved in the production of compounds with specific
biological functions. The one reference to the potential catabolic role of AADCs
in mammalian liver and kidney describes that after oral administration of tyrosine,
tryptophan or phenylalanine to rats the plasma levels for these amino acids
increase (20). When AADC inhibitors are added with the amino acids the plasma
33
levels are increased 2 fold. The resulting amines from these amino acids are
excreted in the urine. AADC is therefore involved in the excretion of surplus
amino acids as their corresponding amines, rather than the complete catabolism
of these amino acids.
Taxa-4(5), 11(12)-diene Synthase
The anticancer compound taxol is biosynthesized from the diterpenoid
skeleton called taxa-4(5), 11 (12)-diene in Taxus brevifolia,. Taxa-4(5), 11(12)- ■
diene is biosynthesized from geranylgeranylpyrophosphate (see figure 15) (71).
The enzyme that catalyzes this reaction, taxa-4(5), 11 (12)-diene synthase, has
been purified and characterized from the yew tree (72). P. raistrickii isolate
H10BA2 also produces taxol and it was assumed that this fungus also contained
a taxa-4(5), 11 (12)-diene synthase to synthesize taxol.
Identification of taxa-4(5), 11(12)-diene synthase activity is determined by
GC-MS analysis of organic extracts of enzyme assays (72). The taxadiene
product can be identified by the elution time from the gas chromatograph and
fragmentation pattern from mass spectral analysis with comparison to a
taxadiene standard. Phosphatase was another enzyme that could compete with
taxa-4(5), 11 (12)-diene synthase for substrate (see figure 16). These enzyme
activities can be differentiated by GC-MS analysis of organic extracts of enzyme
assays. Once it has been shown that enzyme activity is from taxa-4(5), 11(12)-
34
Figure 15. Biosynthesis of taxol from geranylgeranylpyrophosphate and taxa4(5), 11(12)-diene.
Geranylgeranylpyrophosphate
n
?'
?'
&
&
0 — p —o —P—O"
Pyrophosphate
Taxa-4(5), 11(12)-diene
Synthase
0
V *
0
"
HO— P—O—P—O"
& &
Taxa-4(5), 11(12)-diene
TAXO L
"
35
Figure 16. Comparison of phosphatase and taxa-4(5), 11(12)-diene synthase
reactions.
Geranylgeranylpyrophosphate
0
"
0
'
0 — P— 0 — P— 0"
&
&
Pyrophosphate
O
Phosphatase
0"
HO— P—0 —P - O
Il
Il
Geranylgeraniol
OH
Geranylgeranylpyrophosphate
?
'
?
'
0 — p — o — P— 0"
O
Pyrophosphate
Taxa-4(5), 11(12)-diene
Synthase
0'
,
0"
HO— P—0 —P - O
Il
Il
O
O
Taxa-4(5), 11(12)-diene
O
36
diene synthase, and not from phosphatase, the enzyme activity could be
quantified by liquid scintillation counting of organic extracts when all trans
geranylgeranylpyrophosphate [1-3H] was utilized as substrate.
37
CHAPTER 2
AROMATIC AMINO ACID DECARBOXYLASE PURIFICATION AND
CHARACTERIZATION
Introduction
This chapter outlines the materials and methods used to purify and
characterize an aromatic L-amino acid decarboxylase (AADC) from Penicillium
raistrickii isolate H10BA2, as well as the determination of AADC activity in P.
raistrickii ATCC#48678. The results of the purification and characterization of an
AADC from P. raistrickii isolate H10BA2 are presented.
Materials and Methods
Funoal Culture
The fungus designated as H10BA2 was isolated from the inner bark of a
yew tree from the Hungry Horse district of Montana (18). H10BA2 was identified
as a P. raistrickii at the International Mycological Institute by Dr. Zofia Lawrence.
38
The fungus was maintained on IV11DY (M1D [73]+ 1% yew needles) plates. For
the production of AADC1three 1 cm square agar plugs infiltrated with sporulating
mycelium were added to 500 ml of autoclaved (121 °C, 15 psi for 45 min) M1S
media (60 g/l sucrose, 5 g/l Bacto-soytone, and 1 g/l Bacto-yeast extract) in a 2I
erlenmeyer flask. Cultures were incubated as still cultures for 6 days at ambient
temperature and light.
Buffers
Buffers were prepared with reagent grade chemicals and the pH values
were adjusted with hydrochloric acid or sodium hydroxide. The following buffers
were used: wash buffer A (1 mM disodium EDTA10.9% sodium chloride, 10 mM
2-mercaptoethanol, 20 mM sodium acetate, pH 5.0), wash Buffer T (10 mM
disodium EDTA10.9% sodium chloride, 10 mM 2-mercaptoethanol, 20 mM Tris1
pH 7.6), extraction buffer A (10 mM 2-mercaptoethanol, 10 p.M pyridoxal -S 'phosphate, 50mM sodium acetate, pH 5.0), extraction buffer T (1 mM disodium
EDTA, 10 mM 2-mercaptoethanol, 50 mM Tris1 pH 7.6), dialysis buffer A (1 mM
dithiothreitol, 50 mM sodium acetate, pH 5.5), assay buffer (1 mM dithiothreitol, 1
pM pyridoxal-5'-phosphate, 50 mM sodium acetate pH 5.5), and dialysis buffer T
(50 (j,M Pxy-P, 5 mM dithiothreitol, 50 mM Tris1pH 7.6). Chromatography buffers
were based on a solution of 1 mM dithiothreitol, 1 p.M pyridoxal-5'-phosphate, 50
mM sodium acetate, pH 5.0 (Buffer C). Hydrophobic interaction chromatography
(HIC) buffers varied in the concentration of ammonium sulfate added to buffer C.
39
HIC buffers 1, 0.8, 0.6 and 0.0, contained 1.0 M, 0.8 M, 0.6 M and 0.0 M
ammonium sulfate, respectively. Cation exchange chromatography (CAT)
buffers vary in the concentration of potassium chloride added to buffer C. CAT
buffers 100, 120, 140 and 200 contained 100 mM, 120 mM, 140 mM and 200
mM potassium chloride, respectively.
Enzyme Harvest
After six days of incubation, the medium was decanted and the mycelium
patted dry between paper towels. The weight of the “dry” mycelium was
measured. All subsequent steps were performed at 4 °C.
Mycelium was
immersed in 2I of wash buffer, and squeezed dry. The mycelium was washed
twice in this fashion. Mycelium was squeezed dry and added to 500 ml of
extraction buffer. The mycelium was homogenized in an ice/water bath with an
Omni Homogenizer (32x195 mm saw teeth generator) previously cooled to -2 0
°C, with a cycle of 30 sec on, 1 min off, which was repeated 4 times. The slurry
was centrifuged for 15 min at 22,800 x g and the supernatant saved. The
supernatant was centrifuged for 15 min at 22,800 x g and filtered through 4
layers of Miracloth (Calbiochem).
Protein concentration was estimated by the Bradford method (74) using
bovine serum albumin as a standard.
40
Enzyme Activity vs. Length of Culture
P. raistrickii isolate H10BA2 was grown in 3 different types of media over 20
days to determine when the enzyme was being produced. The media were M1S,
M1G (31.6 g/l glucose, 5 g/l soytone, 1 g/l yeast extract), and M1F (31.6 g/l
fructose, 5 g/l soytone, 1 g/l yeast extract). Two 1 cm square plugs of sporulating
P. raistrickii were used to inoculate each 11Roux flask that contained 200 ml of
medium. Each experiment was done in duplicate. On days 2, 4, 6, 9, 11, 13, 16,
18, and 20, the mycelium from a Roux flask was harvested to determine AADC
activity. Protein was harvested from the mycelium as described above, but the
volumes were reduced to 100 ml wash buffer, and 40 ml extraction buffer. The
mycelium was homogenized as above, but the times adjusted to a cycle of 10
sec on, 50 sec off, and repeated 4 times. Enzyme activity was determined by
liquid scintillation counting analysis.
Enzyme Purification
Hydrophobic Interaction Chromatography. Powdered ammonium sulfate
was added slowly to the crude supernatant at 4°C while stirring, to a final
concentration of 1.0 M ammonium sulfate. This was centrifuged for 15 min at
22,800 x g. The supernatant was applied to a 2.5 x 11.5 cm column of
Pharmacia phenyl-sepharose 6 fast flow, previously equilibrated with HIC buffer
1, at a flow rate of 4 ml/min with pressure applied to the top of the column via a
peristaltic pump. The column was rinsed with 200 ml of HIC buffer 1. The
41
enzyme was eluted by a stepped gradient of HIC buffers 0.8, 0.6, and 0.0 using
200 ml of each solution, and 50 ml fractions were collected. Five ml of each
fraction was dialyzed against HIC buffer 0.0 and assayed for enzyme activity by
thin-layer chromatography analysis.
Cation Exchange Chromatography. Active fractions from hydrophobic
interaction chromatography were combined and dialyzed against buffer C. An
open top 1.5 x 1.6.5 cm column of Pharmacia CM (carboxymethyl) Sepharose
Fast Flow cation exchanger was equilibrated with buffer C. Dialyzed fractions
were loaded onto the column at 4 ml/min. The column was rinsed with 100 ml of
buffer C. The enzyme was eluted by a stepped gradient of CAT buffers 120, 140
and 200 using 100 ml of buffer for each step with collection of 10ml fractions.
Fractions, without previous dialysis, were assayed for enzyme activity by thinlayer chromatography analysis. Active fractions were dialyzed against buffer C
and concentrated by chromatography on a 1 x 6 cm column of Pharmacia CM
(carboxymethyl) Sepharose Fast Flow cation exchanger. The enzyme was
eluted by a stepped gradient of CAT buffers 100 and 200 using 32 ml of buffer for
each step with collection of 4 ml fractions. Fractions, without previous dialysis,
were assayed for enzyme activity by thin-layer chromatography analysis.
Fractions were stored at -20°C.
42
Enzyme Assays
AADC activity was detected qualitatively by thin-layer chromatography and
quantitatively by liquid scintillation counting.
Thin-Layer Chromatography-Based Enzyme Assay. Enzyme assays were
performed in 1.5 ml eppendorf tubes with 400 pl assay buffer, 100 pi protein
extract and 20 pi of 5 mM amino acid solution producing a final substrate
concentration of 200 pM. Assays were incubated in a 36 ° C water bath for 1
hour. Enzyme assays were stopped by the addition of 10OpI of 10% sodium
hydroxide. If phenylalanine or analogs of phenylalanine were used as substrate,
the reactions were extracted three times with 750 pi cyclohexane. If tyrosine or
tryptophan were used as substrate, the reactions were extracted three times with
750 pi ethyl acetate, and centrifuged at 9,390 x g to separate the phases.
Organic extracts were transferred by pasteur pi pet to 1.4 dram glass vials, and
solvent removed in vacuo. Organic residues were dissolved in 10pl of
chloroform:methanol (1:1,v/v) and spotted on Whatman AL SIL G/UV silica gel
plates. Thin-layer chromatograms were developed in chloroform : m ethanol:
14.8 N ammonium hydroxide (7:1.8:0.3). Chromatograms were heated until dry
and sprayed with ninhydrin spray reagent (0.3g ninhydrin in 100 ml 1-butanol, 3
ml glacial acetic acid) and heated to 100 °C. Amines were visualized by heating.
The presence of particular amines was determined by their distinctive color
reaction and migration behavior as compared to standards.
43
Standard amines produced the following results: phenethylamine produced
a purple spot at Rf = 0.86, tyramine produced a brown spot at Rf = 0.45, and
tryptamine produced a spot with a brown edge and yellow center at Rf = 0.52.
Liquid Scintillation Counting-Based Enzyme Assay. Enzyme assays were
performed in 5 ml polypropylene test tubes capped with Fisherbrand SAV-IT
closures. Phenylalanine, L-[14C(U)] was purchased from American Radiolabed
Chemicals, Inc. Tyrosine, L-[14C(U)] and tryptophan, L-[side chain-3-14C] were
purchased from Moravek Biochemicals, Inc. Forty jil of protein fraction was
added to 450 pJ assay buffer and 10 pi of 0.1 p Ci of 14C labeled amino acid/500
pM amino acid, giving a final concentration of 10 pM substrate. Controls were
prepared by combining enzyme and buffer in a test tube, boiled for 1 min, and
cooled. After the addition of substrate, the reaction was stopped by injecting 100
pi of 10% sodium hydroxide through the closures into the assay solution and
vortexing. Tubes were mixed for 10 min, extracted three times with 0.5 ml of
organic solvent and centrifuged briefly to separate the phases. Cyclohexane was
used for the extraction of phenethylamine, and ethyl acetate was used for the
extraction of tyramine and tryptamine. The organic extract solution was
transferred to a 20 ml polypropylene liquid scintillation vial containing 5 ml of
ScintiSafe EconoF. Samples were mixed and counted for 3 min on a Beckman
LS 6000 SC. The linear range of the assay extended to 2.3 pg protein/ml for the
phenyl-sepharose active fraction and 1.3 pg protein/ml of purified enzyme with a
44
1 h incubation period. All assays were conducted under linear conditions in
duplicate to allow direct comparison of treatments within an experiment. Enzyme
activity is reported in nanokatals. One Ratal is one mole per second.
Identification of Enzyme Reaction Product
Gas Chromatography - Mass Spectrometry. Enzyme assays were
performed using the thin-layer chromatography method. After the organic
solvent had been removed in vacuo, 20 pJ of dimethylformamide was added to
the vial, followed by 20 pi of N-(tert-butyldimethylsilyl)-N-methyitrifluoroacetamide
as a derivatizing agent. The derivatization reaction was performed at room
temperature for 1 min and was analyzed by GC-MS (Hewlett-Packard 5890
series Il with a 5971 series mass spectrometer). Samples were separated on a
J&W Scientific, DB-5 column, 30 meters long, with an inside diameter of 0.25 mm
and 0.25 pm film thickness. Gas chromatograph parameters were set with the
injector at 280 °C, detector at 300 °C, initial oven temperature at 150 °C for 3 min
with a ramp of 20 °C/min to 300 0C for 30 sec with helium as the carrier gas at
0.9 ml/min flow rate.
Autoradiography of Thin Laver Chromatogram. Enzyme assays were
performed with 400 pi of 50 pM pyridoxal-5-phophate, 5 mM dithiothreitol, 50 mM
Tris pH 7.6, 10 pi substrate, and 100 pi partially purified enzyme. The fungus was
45
grown as described above, except the culture length was 21 days. Enzyme was
harvested from the mycelium in the same manner described above, except wash
buffer T and extraction buffer T were used. Ammonium sulfate precipitation was
performed on the supernatant, and the 50 to 75 % ammonium sulfate fraction
was used as the enzyme source after dialysis against dialysis buffer T.
Ammonium sulfate fractionation was performed by adding powdered ammonium
sulfate to the crude supernatant over 20 min and stirring for 30 min. Precipitated
proteins were removed by centrifugation for 10 min at 10,844 x g. Protein pellets
were resuspended in 1mM dithiothreitol, 0.1 mM EDTA, 50 mM Tris1 pH 7.6 and
dialyzed against this buffer. 14C-PhenyIaIanine was used as the substrate, with a
final concentration of 100 pM phenylalanine (0.4 p C i/100 pM). Organic solvent
was transferred by pasteur pipet to 1.4 dram glass vials and removed under a
stream of nitrogen gas. The residue was subjected to thin-layer chromatography
as previously described. After the thin-layer chromatogram was sprayed with
ninhydrin spray reagent and amines visualized, it was overlaid with Fuji RX
medical x-ray film. The film was exposed for 6 days and developed according to
manufacturer’s specifications.
AADC Utilization of L- or D-Phenvlalanine
Experiments were performed to determine the specificity of fungal AADC for
the L- and D- stereoisomers of the amino acid substrates. Enzyme activity was
determined by thin-layer chromatography-based enzyme assays. Stock
46
substrate solutions contained 5 mM L- or D-phenylalanine. Enzyme assays
contained 100 pi of the phenyl-sepharose active fraction, 380 pi assay buffer,
and 20 pi stock substrate solution. Boiled controls were performed. Assays
were incubated at 34°C for 1 h.
Product Formation vs. Length of Enzyme Assay Incubation
Assays were performed to determine the linear time period for product
formation under optimal conditions. Enzyme activity was determined by liquid
scintillation counting-based enzyme assays. Enzyme assays of the phenylsepharose active fraction contained 75 pi of protein, 415 pi assay buffer, and 10
pi tryptophan substrate to give 0.1 pCi at 10 pM. Duplicate assays were
incubated for 10, 20, 30, 45, 60, 75, and 120 min at 36°C. Enzyme assays of the
purified enzyme contained 60 pi protein, 430 pi assay buffer, and 10 pi
tryptophan substrate to give 0.1 pCi at 10 pM. Duplicate assays were incubated
for 10, 30, 45, 60, 90, and 120 min at 32°C. Boiled controls were performed and
plotted as 0 min.
Optimization of Temperature
Enzyme activity was determined by liquid scintillation counting-based
enzyme assays. Enzyme assays contained 40 pi purified enzyme, and 460 pi
assay buffer, and 10 pi tryptophan substrate to give 0.1 pCi at 10 pM. Assays
were performed at 10, 20, 28, 36, 42, and 50° C in triplicate for 45 min.
47
Optimization of pH
Enzyme activity was determined by liquid scintillation counting-based
enzyme assays. Sodium acetate and citrate/phosphate (75) based buffer
systems were used with 50 mM buffer, 1 mM dithiothreitol, and 1 juM Pxy-P.
Sodium acetate buffers included the following pH values: 5.0, 5.2, 5.4, 5.6.
Citrate/Phosphate buffers included the following pH values: 4.1, 4.74, 5.17, 5.78,
6.26, 6.73, 7.39.
Enzyme assays contained 60 [i\ purified enzyme, 10 |il
tryptophan substrate to give 0.1 ^iCi at 10 jJVl, and 430 \i\ buffer. Assays were
performed in triplicate at 32° C for 45 min.
Optimization of Cofactor Concentration
Pyridoxal-5-phosphate (Pxy-P) is a cofactor utilized by other AADCs to
catalyze the decarboxylase reaction. Experiments were performed to determine
the effect of adding exogenous Pxy-P to enzyme assays. Fifteen ml of the
phenyl-sepharose active fraction was dialyzed extensively against 11of dialysis
buffer with 3 changes of dialysis buffer to remove non-bound Pxy-P. Enzyme
activity was determined by liquid scintillation counting-based enzyme assays.
Enzyme assays contained 100 ^il protein fraction, 380 pJ of the dialysis buffer, 10
pi tryptophan substrate to give 0.1 pCi at 10 pM, and 10 pi of Pxy-P standard to
give desired concentration. Protein, buffer and Pxy-P were combined and
allowed to incubate for 10 min at 36 °C prior to the addition of the substrate and
continued incubation at 36 0C for 30 min.
48
Inhibitors of AADC
The differential effect of the suicide inhibitors L-a-fluoromethyl tyrosine and
L-a-fluoromethyl (3,4 dihydroxyphenyl)-alanine on AADCs from different sources
has been described (76). Experiments were performed to determine the effect of
these inhibitors on the AADC from P. raistrickii isolate H10BA2, thereby allowing
comparison to the reported effects on other AADCs. The AADC inhibitors L-afluoromethyl tyrosine and L-a-fluoromethyl (3, 4 dihydroxyphenyl)-alanine were a
gift from Dr. William L. Henckler at Merck Research Laboratories. Enzyme
activity was determined by liquid scintillation counting-based enzyme assays. A
preincubation was performed with 75 pi of phenyl-sepharose dialyzed active
fraction, 10 pi 1.0 mM inhibitor, and 405 pi assay buffer for 30 min at 36°C. Ten
pi of substrate was added to give a final concentration of 0.1 pCi per 100 pM
tryptophan.
SDS-PAGE
.
SDS-PAGE analysis was performed according to the method of Laemmli
(77). Gels were made with a 4% acrylamide stacking gel and a 6% acrylamide
separating gel in a 1.5 mm thickness. Reducing SDS-PAGE analysis was
performed in the presence of 262 mM 2-mercaptoethanol. Gels were stained
with silver stain (78).
49
Isoelectric Focusing to determine p|
A NOVEX Xcell Il Mini-Cell was used with NOVEX IEF pH 3-7 gels and
buffer system. One lane was used for 8 pl Bio-Rad Laboratories IEF Standards,
pi range 4.45-9.6. The other lanes were loaded with 12.5 pi protein at 15.5 pg/pl
from the phenyl-sepharose dialyzed active fraction of an enzyme harvest, diluted
1:1 with NOVEX IEF sample buffer. Gels were focused for I h at 100V, 35 min
at 200V and 40 min at 300V. The gel was removed from the cassette and the
standard lane stained with coomasie blue. The rest of the gel was sliced
perpendicular to the pH gradient in 3 mm wide slices. Gel slices were prepared
for determination of enzyme activity by liquid scintillation counting analysis.
Slices were transferred to test tubes containing 1 ml of 4° C assay buffer and
kept at 4°C for 1 hour. Buffer was removed by aspiration and the gel Crushed
with a spatula. Five hundred pi of assay buffer and 10 pi of 0.1 pCi /SOOpM
tryptophan were added. Assays were incubated at 36°C for 1 hour.
Determination of Km for Substrates
Enzyme activity was determined by liquid scintillation counting-based
enzyme assays. Enzyme assays used 100 pL of the phenyl-sepharose dialyzed
active fraction of an enzyme harvest, 10 pL substrate with 0.1 pCi of radiolabeled
substrate for each substrate concentration, and 390 pL of assay buffer. Assays
were performed in triplicate at 36° C for 45 min and stopped by placing into
50
boiling water for 1 min. The Km values were determined by plotting the average
of each substrate concentration as Lineweaver-Burk reciprocal plots.
Detection of AADC activity in P. raistrickii ATCC#48678
Experiments were performed to determine if AADC activity was present in
another isolate of Penicillium raistrickii grown under identical conditions utilized
for isolate H10BA2. Penicillium raistrickii ATCC # 48678 was chosen because it
also was isolated from a plant source and resembled isolate H10BA2 when
grown on identical media. Penicillium raistrickii ATCC # 48678 was grown in
M1S media and the enzyme activity determined in the same manner as cultures
in the enzyme activity vs. length of culture experiments with tryptophan as
substrate. Mycelium was harvested on days 7 and 17. Two Roux flask cultures
were harvested for each day. Enzyme assays were performed in duplicate for
each culture. Enzyme activity was determined by liquid scintillation countingbased enzyme assays.
Results
Identification of Enzyme Reaction Product
AADC activity was detected in the soluble fraction of the mycelial
homogenate. This activity was detectable by thin-layer chromatography analysis
of the enzyme reaction when L-phenylalanine, o-fluoro-D,L-phenylalanine, p-
51
fluoro-D,L-phenyla!anine, L-phenylalanine methyl ester, L-tyrosine and Ltryptophan were used as substrates as shown in figure 17. These amino acids
were utilized to form the corresponding amines o-fluorophenethylamine (A),
phenethylamine (B), p-fluorophenethylamine (C)1tryptamine (D) and tyramine (E)
with the correct Rf and color reaction with ninhydrin spray compared to standard
amines. Amino acids that did not produce detectable levels of amines were L-5hydroxytryptophan, and L-histidine.
The autoradiography of a thin-layer chromatogram of the organic extracts of
enzyme assays performed with 14C-IabeIIed phenylalanine as substrate was
performed to demonstrate that the amine product could be separated from the
amino acid substrate by extraction under basic condition (see figure 18). Lanes
1, 2 and 3 contained the organic extracts of enzyme assays performed for 15, 30
and 45 minutes respectively. Two amines, designated A and B, were present in
lanes 1, 2 and 3, after using a ninhydrin spray reagent to visualize amines.
Amine A traveled at the solvent front and was an artifact from the enzyme
solution since it was present in the organic extracts of boiled enzyme control
shown in lane 4. Amine B had the same Rf and color reaction as the
phenethylamine standard in lane 5. The autoradiogram of the thin-layer
chromatogram demonstrated that amine B was radiolabeled, thereby indicating
that it was derived from the 14C-IabeIed phenylalanine. The ninhydrin spray
analysis of the thin-layer chromatogram and the corresponding autoradiogram
demonstrated that there was no amino acid substrate present in the organic
52
Figure 17. Thin-layer chromatography analysis of enzyme products. Assays
were performed with partially purified enzyme from phenyl-sepharose
chromatography, using 0.2 mM substrate as described in materials and methods.
Substrates used were lane 1, L-phenylalanine; 2, o-fluoro-D,L-phenylalanine; 3,
p-fluoro-D,L-phenylalanine; 4, L-phenylalanine methyl ester; 5, L-tyrosine; 6,
Blank; 7, L-tryptophan; 8, L-5-hydroxytryptophan; 9, L-histidine, 10, boiled
enzyme and L-tryptophan. Amines produced in lanes 1, 2, 3, 4, 5 and 7
correspond with A, o-fluorophenethylamine; B, phenethylamine; C, pfluorophenethylamine; D, tryptamine and E, tyramine.
V
V
9
'7
i
A
mO
D
#
E
1
2
3
4
5
6
7
8
9
10
f
Figure 18, Comparison of a thin-layer chromatogram and the corresponding autoradiogram of the reaction
products of an aromatic amino acid decarboxylase assay. Enzyme assay was performed with 14C (U)phenylalanine as substrate. Figure A is the thin-layer chromatogram of the reaction products developed as
described in the materials and methods. Figure B is the autoradiogram of figure A. Lanes 1 through 3 contained
enzyme reaction products extracted from assays performed for 15, 30, and 60 min, respectively. Lane 4 contained
the extractable compounds from a boiled enzyme control incubated for 60 min. Lane 5 contained an unlabeled
standard of phenethylamine. Lane 6 contained a 14C(U)-phenylalanine standard. A = unidentified amine from
enzyme assay, B = phenethylamine, C = phenylalanine, D and E = contaminants of 14C(U)-phenylalanine.
Figure A. Thin-layer chromatogram of reaction products
1
2
3
4
5
6
Figure B. Autoradiogram of Figure A
1
2
3
4
5
6
54
extracts of the enzyme assays in lanes 1 , 2 , 3 and 4, because they lacked a spot
corresponding to the same Rf as the phenylalanine standard in lane 6. The
phenylalanine standard in lane 6 contained other compounds, designated D and
E, that did not contain amine functional groups that reacted with the ninhydrin
spray. Compounds D and E are considered to be radiolabelled contaminants of
the phenylalanine substrate. Compound D was coextracted with phenethylamine
in lane 3, and by comparing the relative intensities of the spots did not contribute
a significant amount of radioactivity.
GC-MS analysis of an enzyme reaction with tryptophan as substrate yielded
the decarboxylated product tryptamine, which had the same elution time and
fragmentation pattern as the standard, as shown in figures 19 and 20.
AADC Utilization of L- or D-Phenvlalanine
Fungal AADC specificity for the L- and D- stereoisomers of phenylalanine
was determined. Figure 21 demonstrated that fungal AADC produced
phenethylamine when incubated with L-phenylalanine by the presence of a spot
(B) in lane 5 that had the same Rf and color reaction as the phenethylamine
standard in lane 7. No detectable phenethylamine was present in lane 6 when
AADC was incubated with D-phenylalanine under identical conditions as lane 5.
The amines present at the solvent front (A) were artifacts extracted from the
enzyme buffer solution since they were present in the boiled enzyme control 3, 4,
5 and 6 demonstrated the efficacy of organic solvent extraction of basic enzyme
Figure 19. Mass spectrum of derivatized tryptamine extracted from an aromatic amino acid decarboxylase assay
and separated on a gas chromatograph as described in the materials and methods.
/^serage of 9.734 to 9.815 rrin.:
/N D L rx d a n o e
130
8500
8000
7000
6500
6000
Ol
Ol
5500
5000
H2C = N -S i-C -C H 3
4000
hJ
="=
3500
3000
2500
2000
103
1500
IOOO
115
102
500
188
160
100
120
140
180
200
Figure 20. Mass spectrum of a derivatized tryptamine standard separated on a gas chromatograph as described in the
materials and methods.
Average of a 769 to 9.801 im : BRD93D.D ( t l
Abirdance
5000
H2C = N -S i-C -C H 3
hH3C - 3
217
m fz-y
100 120
H
57
Figure 21. Thin-layer chromatography analysis of phenethylamine production in
enzyme assays utilizing L- or D-phenylalanine as substrate. L a n e l, Lphenylalanine standard; Lane 2, D-phenylalanine standard; Lane 3, Boiled
enzyme + L-phenylalanine; Lane 4, Boiled enzyme + D-phenylalanine; Lane 5,
assay of phenyl-sepharose active fraction with L-phenylalanine; Lane 6, assay of
phenyl-sepharose active fraction with D-phenylalanine; Lane 7, phenethylamine
standard. A = unidentified amine from enzyme assay buffer, B =
phenethylamine, C = phenylalanine.
Solvent
Front
assay solutions to separate the amine from the amino acid. The AADC isolated
from P. raistrickii isolate H10BA2 was specific for the L-isomer of the amino acid
substrate.
58
Product Formation vs. Length of Enzyme Assay Incubation
Enzyme assays were performed with varying lengths of incubation to
determine the linear range of the assay for the phenyl-sepharose active fraction
and purified enzyme. Figure 22 is a plot of the concentration of tryptamine
produced by the phenyl-sepharose active fraction at each time point. Figure 23
is a plot of the concentration of tryptamine produced by purified enzyme at each
time point. These figures demonstrated that the linear range of the enzyme
assays was between 10 and 60 min. This meant that the velocity of the enzyme
reaction remained constant through this time period and that assays performed
within this time period were comparable.
Figure 22. Concentration of tryptamine produced by phenyl-sepharose active
fraction at different times of incubation. Averaged duplicate assays ± one
standard deviation.
y = 0.027x + 0.1011
tL
0.5
R2 = 0.9889
59
Figure 23. Concentration of tryptamine produced by purified enzyme at different
times of incubation. Averaged duplicate assays ± one standard deviation.
0.6 n
^
0.5 -
aT 0.4 § 0.3 > ?
0.2
-
t O,o l0
Enzyme Activity vs. Length of Culture
Enzyme activity was present from day 4 until time of harvest in the three
different media, using three different carbon sources, as seen in figure 24. The
three carbon sources were sucrose, glucose and fructose. Comparison of
specific enzyme activity in the mycelia grown on sucrose to the mycelial wet
weight is presented in figure 25. The peaks of high specific enzyme activity per
gram mycelial wet weight for each treatment are shown in table 1.
Table 1. Peaks of high specific enzyme activity per gram mycelial wet weight
from mycelia grown in three different carbon sources. Values are in (nkatal/mg
protein)/ gram mycelial wet weight.
Treatment
Sucrose
Glucose
Fructose
Day 4
Day 18
19
13
17
9
12
18
Figure 24. Aromatic amino acid decarboxylase activity from mycelia of P. ra istrickii grown in three different media
during a 20 day culture is on the y-axis. Media differed in carbon sources that included sucrose, glucose and
fructose. Average of duplicate assays ± 1 standard deviation.
Sucrose
- O - Glucose
Fructose
200
2
160
E 140
5
120
100
Day of Culture
Figure 25. Mycelial “wet” weight compared to aromatic amino acid decarboxylase activity from mycelia of P.
raistrickii grown in MIS medium during a 20 day culture is on the left y-axis. Average of duplicate assays. The
average mycelial “wet” weight of all treatments in the three different media (see figure 21) at time of harvest is on
the right y-axis ± 1 standard deviation.
#- Sucrose
4 3 - Mycelial Wet Weight
140
§
10.00
120
0
CL
8.00
E 100
1
CO
JxC
C
80
6.00
I
F
%
4.00
I-
OJ
£
IT
40
CQ
0
Q
1
2.00
20
0.00
Day of Culture
62
Purification of Aromatic L-Amino Acid Decarboxylase
Enzyme activity was determined throughout the purification steps by thinlayer chromatography based enzyme assays. Enzyme activity was found in the
0.6M ammonium sulfate fraction from the phenyl-sepharose chromatography and
in the 140mM potassium chloride fraction from the cation exchange column.
Purity of fractions was determined by SDS-PAGE analysis. The 140mM
potassium chloride fraction was concentrated by a subsequent cation exchange
chromatography step. Enzyme activity was found in the 200mM potassium
chloride fraction. The 20.0mM potassium chloride fraction contained one protein
band with a molecular weight of 125,000 Da ± 3,000 by silver staining (see figure
26). The recovery from one harvest of aromatic amino acid decarboxylase is
presented in table 2. The specific activity dropped between the phenylsepharose and the CM-sepharose purifications. This drop in specific activity
could be from the loss of enzyme activity in the CM-sepharose active fraction by
repeated freeze thaw cycles. Table 3 demonstrated that specific activity of the
purified enzyme from CM-sepharose active fraction did decrease from repeated
freeze thaw cycles.
Table 2. Purification of aromatic amino acid decarboxylase from one
purification procedure.
Purification Step
Total Protein
Specific Activity
% Recovery
(nkatal/mg)
(mg)
Soluble Supernatant
262.26
552
TOO
Phenyl-Sepharose
3.52
26183
64
CM-Sepharose
0.14
10455
1.0
63
Figure 26. Non-reducing SDS-PAGE (6% polyacrylamide) analysis of enzyme
purification steps. Lane 1, crude supernatant; Lane 2, Active fraction after
phenyl-sepharose purification step; Lane 3, Active fraction after cation exchange
purification and concentration step. Molecular weight standards are indicated in
205
125
116
97
64
Table 3. Decreased specific activity in purified enzyme by freeze/thaw cycle
between two subsequent experiments of enzyme activity.
Test for Enzyme Activity
,
Specific Activity
(nkatal/mg)
From: Effect of Temperature on
5618
Enzyme activity. Figure 27
From: Effect of pH on Enzyme
3023
activity. Figure 28
Properties of Aromatic L-Amino Acid Decarboxylase
The effect of temperature (figure 27) and pH (figure 28) on enzyme activity
was determined. The temperature for maximum enzyme activity was between
28° and 36°C (see figure 27). The pH for maximum enzyme activity was
between pH 5.0 and 5.6 (figure 28). Addition of Pxy-P did not have a significant
effect on enzyme activity (see figure 29). The Km values for L-tryptophan, Lphenylalanine, and L-tyrosine were determined by double-reciprocal plots (see
figures 30, 31 and 32, respectively). The isoelectric point of fungal AADC was
determined by isoelectric focusing gel electrophoresis. Enzyme activity was
limited to the pH range of 6.2 to 6.4 (figure 33) in an isoelectric focusing gel
indicating that the enzyme has a pi within that range. The protein subunit
structure for fungal AADC was determined by reducing and non-reducing SDSPAGE analysis of purified protein (see figure 34). Fungal AADC yielded a single
band with a molecular weight of 125,000 ± 3,000, as estimated under
nonreducing SDS-PAGE conditions (see figure 34, Iane3). Under reducing SDSPAGE conditions, there was still one band present with a molecular weight of
125,000 (see figure 34, lane 2). This indicates that AADC is a monomer. A
65
summary of the characteristics of fungal AADC and AADCs isolated from other
sources are presented in table 4.
Figure 27. Effect of temperature on the activity of pure aromatic amino
acid decarboxylase. Averaged triplicate assays ± one standard deviation.
S
2.5
o
0.5
Temperature, C
Figure 28. Effect of pH on purified AADC activity measured in two different
buffers, citrate/phosphate (closed circle) and acetate (open circle).
Averaged triplicate assays ± 1 standard deviation.
66
Figure 29. Effect of pyridoxal phosphate concentration on the activity of
extensively dialyzed aromatic amino acid decarboxylase from phenylsepharose active fraction. Averaged duplicate assays ± one standard
deviation.
r
16
14
e
12 8
i° >
8
6
4
2
1.00E-09 1.00E-07 1.00E-05
<:
=3
£ti
O
£"
=3
1.00E-03
[Pyridoxal 5-Phosphate], M
Figure 30. Double reciprocal plot of hydrophobic interaction chromatography
active fraction with tryptophan as substrate.
0.06
y = 0 .3 8 7 1 X + 0 .0 0 8
R2 = 0 .9 9 7 3
Km = 48.4 pM
Vmax = 125 nIWmin
-1/Km
1/Vmax
-0.03
2
0.07
0.
1/[Tryptophan], (IVI)-I x 10-6
67
Figure 31. Double reciprocal plot of hydrophobic interaction chromatography
active fraction with phenylalanine as substrate.
<?
O
0.008
^
0.006
y = 0.0594x + 0.0006
R2 = 0.9977
Km = 99 pM
^
Vmax = 1666 nM/min
■|
0.004
X
I,
0.002 -
r~
-0.02
I
I
0.03
0.08
I
0.13
1/[Phenylalanine], (M)-1 x 10-6
Figure 32. Double reciprocal plot of hydrophobic interaction chromatography
active fraction with tyrosine as substrate.
a)
O
0.1
x
0.08 -
I
V
C
0.06 -
x
0.04 -
T
g
0.02
-0.02
y = 0.7485x + 0.0007
R2 = 0.9955
Km = 1064 pM
Vmax = 1422 nM/min
0.03
0.08
1/[Tyrosine], (M)-1 x 10-6
0.13
68
Figure 33. Aromatic amino acid decarboxylase activity in gel slices from the
isoelectric focusing electrophoresis experiment for the determination of the
isoelectric point.
0
5
10
Gel Slice Number
15
69
Figure 34. SDS-PAGE (6% polyacrylamide slab gel) analysis of aromatic amino
acid decarboxylase with silver stain technique. Lane 1, molecular weight
standards; Lane 2, AADC under reducing conditions; Lane 3, AADC under non­
reducing conditions.
I
2
Molecular
Weight
Standards
(kDa)
205
70
Table 4. Comparison of aromatic amino acid decarboxylase from different sources.
Source (reference)
Substrate, Km QJVl)
pH,
M.W.
Pi
assay
(Da)
optimum
Fungal
Penicillium raistrickii
Trp, 48, Phe, 99, Tyr, 1x103 5.0 - 5.6 125,000 6.2
to
Mammalian
6.4
Human
pheochromocytoma (1)
DOPA, 46, 5-HTP, 67
7.0
100,000 5.7
Rat Liver (2)
DOPA186
6.9
100,000 5.7
Hog Kidney (3)
DOPA, 190, 5-HTP, 100
8.5
112,000 NR
Trp,1x104, Phe, 4.2x104
Tyr, 8.4x103
Guinea Pig Kidney (5)
DOPA, 400, 5-HTP, 20
9.0
NR
NR
Trp, 3x1031Phe, 2x104
Tyr, 1.3x104
Bovine Brain (4)
DOPA, 140, 5-HTP,120
7.2
100,000 4.9
TO
5.3
Plant
Phalaris tuberosa (J)
Catharanthus roseus (10)
Eschscholtzia californica
(11)
Thalictrum rugosum (11)
Trp, 200
Trp, 75, 5-HTP, 1.3x103
Tyr, 250, DOPA, 1.1X103
7.6
8.5
8.4
NR
115,000
112,600
NR
5.9
5.2
Tyr, 270, DOPA, 240
8.4
112,600
5.4
Fish
Skipjack tuna liver (12)
5-HTP, 697
7.0
110,000
NR
Insect
Calliphora vicina (13)
DOPA, 280
6.8
9096,000
NR
Bacteria
Streptococcus faecalis
Tyr, 600
>200,000
5.5
(14,15)
Trp = L-tryptophan, Tyr=L-tyrosine, Phe = L-phenylalanine, DOPA= L-3,4dihydroxyphenylalanine, 5-HTP = L-5-hydroxytryptophan, NR = not reported
4.5
71
Effect of Inhibitors on Aromatic L-Amino Acid Decarboxylase
The effects of L-a-fluoromethyl tyrosine and L-a-fluoromethyl(3,4 dihydroxyphenyl)alanine on fungal AADC activity, and reported effects on other AADCs are
presented in table 5. There was no effect on enzyme activity by either inhibitor.
Table 5. Comparison of effects of a-fluoromethyl analogues on aromatic
amino acid decarboxylase activity from different sources.
Source (reference)
% Inhibition by 20 pM analogue after
preincubation.
a-fluoromethyltyrosine
a-fluoromethylDOPA
PeniciHium raistrickii
-2
3
Syringa cell cultures (76)
Nicotiana cell cultures (76)
Hordeum roots (76)
Sanguinaria rhizomes (76)
Streptococcus cells (76)
12
12
26
0
96
8
3
94
0
89
Detection of AADC activity in P. raistrickii ATCC#48678
AADC assays were performed on crude protein preparations from cultures
of P. raistrickii ATCC#48678 grown for 7 and 17 days in the same manner
performed for P. raistrickii isolate H10BA2. AADC activity was not detectable in
the crude protein preparations from cultures of P. raistrickii ATCC# 48678, grown
for 7 and 17 days.
72
Discussion
The investigation of AADC activity in the fungus Penicillium raistrickii
actually began as a search for the enzyme phenylalanine-2, 3-aminomutase.
Phenylalanine-2, 3-aminomutase is an enzyme that may theoretically participate
in the biosynthesis of the N-benzoylphenylisoserine side chain of taxol. The
reaction catalyzed by this enzyme activity would be the transfer of the amino
group of a-phenylalanine from the a - or #2 carbon to the (3- or #3 carbon,
resulting in the formation of p-phenylalanine (figure 35). The participation of
phenylalanine-2, 3-aminomutase in the biosynthesis of the phenylisoserine side
chain of taxol was demonstrated by Dr. Paul Fleming et. al. after performing
feeding studies with deuterated phenylalanine analogs in yew cultures (79). P.
raistrickii isolate H10BA2, produces taxol and we wanted to see if phenylalanine2, 3-aminomutase was involved in the production of fungal taxol. While
developing an assay and looking for phenylalanine-2, 3-aminomutase activity in
protein extracts of P. raistrickii isolate H10BA2, it was discovered that
phenethylamine was being produced. This indicated that an AADC was present
in this fungus.
Figure 35. Reaction performed by phenylalanine-2, 3-aminomutase.
73
A review of the literature on aromatic amino acid decarboxylases showed
that this class of enzymes is found in a variety of organisms and has a role in the
biosynthesis of natural products. But this class of enzyme had not been
described from a fungal source. Therefore, this enzyme activity from a fungal
source was isolated and characterized.
To begin the investigation of an enzyme, two things are critical: a source of
enzyme and the development of an assay for enzyme activity. P. raistrickii was a
good source for enzyme because it could be grown under defined conditions in
large batches utilizing basic microbiological culture techniques whenever enzyme
was required. P. raistrickii also produces a range of biologically active
compounds derived from aromatic amino acids (see figure 1). It is possible that
AADC functions in the biosynthesis of one or more of these natural products.
The presence of the enzyme was determined by assaying for enzyme
activity. A general assay involves the combination of a protein solution to a
suitable buffer and substrate, which is incubated at a suitable temperature for a
specific length of time. As described in the materials and methods the enzyme
assay for AADC activity was stopped by the addition of 10% sodium hydroxide.
The addition of sodium hydroxide resulted in an increase of pH to 12. Sodium
hydroxide reacts with aqueous carbon dioxide to form sodium bicarbonate. This
acted to tra p 14C02 that was produced when utilizing uniformly labeled amino
acids. The increase in pH to 12 also allowed for the separation of amino acid
substrates from the amine products by extraction with organic solvents. At basic
74
pH values the carboxylic acid group of an amino acid is deprotonated, and the
'
amine group is neutral giving the molecule a net negative charge. This made the
substrate molecule hydrophilic and unable to be extracted from the enzyme
assay solution by hydrophobic organic solvents. The amine group on the amine
product has no charge at a basic pH, thereby allowing the amine product to be
extracted from the enzyme assay solution by organic solvent. Analysis for the
presence of the amine product in the organic extracts of enzyme assays was
performed qualitatively by thin-layer chromatography or gas
chromatography/mass spectrometry and quantitatively by liquid scintillation
counting. Separation of the amino acid substrate from the amine product by
organic extraction of basic enzyme assay solutions was demonstrated by the
thin-layer chromatography analysis of organic extracts of enzyme assays shown
in figure 18.
To determine the range of amino acids that AADC from P. raistrickii can
utilize as substrate, the organic extracts of enzyme assays with different amino
acids as substrates were analyzed by thin-layer chromatography (see figure 17).
As demonstrated in figure 17, amines were produced by enzyme activity when Lphenylalanine, o-fluoro-D,L-phenylalanine, p-fluoro-D,L-phenylalanine, Lphenylalanine methyl ester, L-tyrosine and L-tryptophan were added as
substrate. The amino acids L-5-hydroxytryptpphan and L-histidine were not
utilized as substrates since no detectable amines were present. DOPA was not
tested as a substrate because dopamine production was not detectable using
75
these methods. Thin-layer chromatography is not a definitive method for the
identification of compounds since only the Rf and reaction with ninhydrin spray
reagent is analyzed. For this reason, products from enzyme assays were
analyzed by gas chromatography/mass spectrometry. Gas chromatography/
mass spectrometry is a definitive technique for identifying compounds by
analyzing the elution time from the gas chromatograph and the fragmentation
pattern produced by mass spectral analysis. Fragmentation patterns, developed
by mass spectral analysis, represent the fingerprint of a compound.
Figure 19
demonstrated that the organic extract of an enzyme assay utilizing tryptophan as
substrate produced a product that had the same elution time and fragmentation
pattern as the tryptamine standard in figure 20. This was definitive proof that
tryptamine was produced by an aromatic amino acid decarboxylase from the
fungus P. raistrickii isolate H10BA2. It could also be inferred that the other
amines demonstrated by thin-layer chromatography analysis of organic extracts
from enzyme assays also contained the appropriate amines.
The ability of the AADC from P. raistrickii isolate H10BA2 to utilize three
naturally occurring amino acids is not unique. The AADC from hog kidney and
guinea pig kidney were reported to utilize DORA, 5-hydroxytryptophan,
tryptophan, phenylalanine and tyrosine (3, 5). Plant AADCs are typically narrow
in their specificity for amino acids (see table 4). The AADCs from Eschscholtzia
californica and Thalictrum rugosum only decarboxylated tyrosine and DORA, and
did not decarboxylate phenylalanine (11), The AADC from Catharanthus roseus
76
decarboxylated only tryptophan and 5-hydroxytryptophan (10). In this respect,
the AADC from P. raistrickii isolate H10BA2 is more similar to mammalian
AADCs.
The “Michaelis” constant, Km, was determined for the substrates
tryptophan, phenylalanine, and tyrosine (see table 4). This constant indicates an
enzyme’s affinity for substrate, the lower the Km the higher the affinity. By this
analysis the AADC from P. raistrickii isolate H10BA2 has a higher affinity for
tryptophan than phenylalanine or tryptophan. Tryptophan is utilized in a variety
of natural products isolated from fungi (see figure 12). AADCs vary widely in
substrate affinity, as seen in table 4 by comparing the Km values of the different
substrates. If enzyme affinity for a substrate is correlated to biological function,
then these differences in substrate affinity can be explained by the corresponding
function of the enzymes in each organism. In mammals and fishes, AADC
functions to make the neurotransmitters dopamine and serotonin; therefore those
enzymes should have a higher affinity for the precursors DOPA and 5-HTP (19).
In insects, AADC functions in the production of melanin from DOPA and could
explain why this enzyme is limited to utilization of DOPA (14). In plants, AADC
functions in the production of secondary metabolites (8, 10, 11), including a
phytohormone (6), and may be involved in plant defenses against pathogens (40,
41). Each enzyme described from a plant has different substrate affinities based
upon the secondary metabolites produced, such as the AADC from Catharanthus
roseus can only decarboxylate tryptophan and tryptophan analogs which feed
77
into indoleamine biosynthesis. From this analysis it is possible that the AADC
from P. raistrickii isolate H10BA2 functions in the production of natural products
derived from tryptamine.
The ability of the AADC from P. raistrickii isolate H10BA2 to utilize the
fluorophenylalanine amino acid analogues is not unique. The Catharanthus
roseus AADC DNA sequence has been used as a selectable marker in
transgenic tobacco cells on 4-methyl tryptophan since it decarboxylated this toxic
tryptophan analogue to the non-toxic 4-methyltryptamine (66). Fluorine is similar
to hydrogen in size, so steric hindrance should not result in exclusion of the
fluorophenylalanine substrates (80). p-Fluorophenylalanine is toxic to fungi and
has been used on Penicillium chrysogenum to induce haploids (81). The toxicity
of o-fluorophenylalanine has not been determined. If the resulting amines from
o- and p-fluorophenylalanine are not toxic, then AADC from this fungus could be
used as a resistance gene, once the gene is isolated.
That the phenylalanine methylester is decarboxylated is in agreement with
the reaction mechanism presented in figure 4. The Pxy-P cofactor does not
interact directly with the carboxylic acid moiety. Progression of the enzyme
reaction would be similar to that shown in figure 4, but would be preceded by the
hydrolysis of the methyl ester to form methanol and phenylalanine in the enzyme
active site. The product being formed may be from phenylalanine formed by
previous hydrolysis of the phenylalanine methyl ester in the enzyme assay buffer
system.
78
To demonstrate that the AADC from P. raistrickii isolate H10BA2 is specific
for the L-isomer, or the naturally occurring amino acid isomer, thin-layer
chromatography analysis of organic extracts of enzyme assays utilizing L- and Dphenylalanine was performed (see figure 21). As demonstrated by figure 21 the
AADC from P. raistrickii isolate H10BA2 is specific for the L-isomer. The ability
of this AADC to utilize only the L-isomer is consistent with the induced fit
hypothesis of enzyme active sites (82). In this hypothesis the substrate induces
the correct three-dimensional arrangement of amino acid R-groups in an enzyme
active site for the reaction to occur. Since the substrate is a three dimensional
structure only the L-isomer would place the amino group in the correct area of
the enzyme active site to react with the aldehyde group of the pyridoxal-5phosphate cofactor (see figure 4). The correct orientation of the L-isomer within
the enzyme active site could be assured by the presence of an arginine or lysine
group present in the enzyme active site that would interact with the carboxylic
acid moiety of the amino acid. Presence of such a group has been suggested by
the site directed mutagenesis studies performed on rat AADC (26). Interaction of
the carboxylic acid moiety of the D-stereoisomer within the enzyme active site
would place the amino group in the incorrect area of the enzyme active site to
react with the aldehyde group of the pyridoxal-5-phosphate cofactor (see figure
36).
79
Figure 36. Demonstration of the proposed difference between L-phenylalanine
and D-phenylalanine, and the spatial relationship between the respective amine
groups to the aldehyde group of pyridoxal-5-phosphate in the enzyme active site.
L-phenylalanine
Il
CH
R
D-phenylalanine
Il
CH
I
R
R = Pyridoxal-5-phosphate moiety
Optimal conditions for the assay of AADC activity from P. raistrickii isolate
H10BA2 were determined by varying the temperature, pH and Pxy-P
concentration. The optimal pH for the enzyme assay ranged from 5.0 to 5.6 in a
sodium acetate based buffer (see figure 28). This value is low relative to the
optimal pH values of the AADCs from other eucaryotic sources, but is similar to
the AADC from Streptococcus faecalis (see table 4). Since the optimal assay pH
is between 5.0 to 5.6, it can be assumed that the enzyme occurs within the
fungal cell in a location where the pH is that value, such as the vacuole. The
optimal pH values of the different AADCs do not affect the charged status of the
respective amino acids since all of the aromatic amino acids exist with both the
carboxylic acid and the amine group in their charged states between pH 5.0 to
9.0.
80
The optimal temperature for the enzyme assay was shown to be between
28 to 36 °C (see figure 27). The fungus probably does not exist at this
temperature outside of the laboratory, but probably occurs at 15 to 25 °C, at
which temperatures the enzyme still functions. That the enzyme activity is
optimal between 28 to 36 0C is most likely attributed to kinetic effects. As
temperature increases the reaction rate increases also until the enzyme is
denatured. This demonstrated that the enzyme performed the reaction from 28
to 36 °C.
As previously mentioned in chapter 1, all of the described AADCs utilize
Pxy-P as a cofactor. It was therefore assumed that the enzyme from P. raistrickii
isolate H10BA2 would also utilize Pxy-P as a cofactor. An attempt to optimize
the pyridoxal-5-phosphate concentration in the enzyme assay solution was made
(see figure 29). These assays utilized partially purified enzyme from a
hydrophobic interaction chromatography preparation. This protein was dialyzed
extensively against a buffer not containing Pxy-P in an attempt to remove Pxy-P
from the enzyme. As demonstrated in figure 29, there was no difference in
enzyme activity between the different treatments lacking and containing Pxy-P.
These results are similar to experiments performed with other AADCs in which
enzyme activity was still present after dialysis. However in reports of other
AADCs, enzyme activity did increase 2 to 3 fold after the addition of Pxy-P to the
assay buffer (3, 4). This increase was not observed for fungal AADC. These
experiments do not support a Pxy-P requirement by this enzyme. To prove that
81
this enzyme utilizes Pxy-P as a cofactor, a sufficient quantity of purified enzyme
would need to be assayed for the presence of Pxy-P by fluorometric techniques
(83). There is a possibility that this enzyme does not utilize Pxy-P as a cofactor
but has some other functional group to perform the reaction. Examples of
enzymes utilizing a pyruvyl moiety were described in chapter 1.
Experiments were performed to determine when enzyme activity was
present throughout the length of a culture (see figures 24 and 25). Figure 24
demonstrated that enzyme activity was detectable from day 4 through day 20.
Two peaks of high specific enzyme activity occurred around days 4 and 18.
Comparison of these peaks to the mycelial wet weight (see figure 25)
demonstrated that these peaks corresponded to the lag and stationary phases of
the culture. This may correspond to utilization of the enzyme in the biosynthesis
of secondary metabolites. Secondary metabolites are typically produced during
the lag and stationary phases of cultures of microorganisms (34). This indicated
a possible function of AADC from P. raistrickii isolate H10BA2 in the biosynthesis
of secondary metabolites. The AADC that was purified and characterized
corresponds to the enzyme activity produced in the first peak of activity at day 4.
It is possible that a different AADC is being produced at the second peak of
activity at day 18. It has been demonstrated that there is a single gene coding
for AADC in both neuronal and non-neuronal of rat and bovine tissues (84).
Opium poppy (Papaversomniferum) contains a gene family with 10 to 14 AADC
82
genes within the genome (36). Members of this gene family are differentially
expressed in various plant tissues.
Preliminary experiments determining enzyme activity during the length of a
culture with 500ml of M1S medium / 2I Erlenmeyer flask indicated that the first
peak of AADC activity occurred around day 6 (data not shown). This time period
was chosen for harvesting enzyme activity because of the relatively high
occurrence of specific enzyme activity per gram mycelia (see table 1).
Purification of AADC to a single protein was accomplished as demonstrated by
SDS-PAGE analysis of the cation exchange chromatography active fraction (see
figure 26). This gel showed that there was only one protein with a molecular
weight of 125,000 Da present after the cation exchange chromatography step.
Further analysis of this purified protein by reducing and non-reducing SDS-PAGE
indicated that this protein was a monomer, since one protein of identical
molecular weight was still present under each treatment (see figure 34).
Reducing SDS-PAGE analysis resulted in the reduction of disulfide bridges that
can covalently link small monomers together to form a large multimer. The
specific activity of the enzyme purification steps (see table 2) also indicated that
the enzyme was being purified as the specific activity increased between the
crude protein fraction and the hydrophobic interaction chromatography steps.
The specific activity decreased between the hydrophobic interaction and the
cation exchange chromatography steps. One reason for a decrease in specific
activity was attributed to a loss of enzyme activity in the purified enzyme fractions
83
from repeated freeze/thaw cycles (see table 3). This demonstrated that the
enzyme is not completely stable under this method of storage. Another reason
for the decrease in specific enzyme activity could be the separation of the
enzyme from an unidentified cofactor or interaction with other proteins. There
are several methods that could be used to identify such a cofactor or protein
interaction. If the cofactor is a small molecule and is stable after boiling, boiled
crude or partially purified protein preparations could be added to the purified
enzyme preparation to see if enzyme activity increased. Boiling crude or partially
purified protein preparations would be required to remove any background
enzyme activity. Another method would entail recombining fractions from the last
chromatography step to identify if there was anything present in them that
increased enzyme activity. It is possible that AADC is associated with a larger
enzyme complex involved in the biosynthesis of secondary metabolites.
Removal of AADC from this complex could result in a decrease of enzyme
activity. Existence of such an enzyme complex could be investigated by
subjecting a crude protein preparation to gel filtration chromatography. Gel
filtration chromatography separates proteins by size. Utilization of a calibrated
gel filtration chromatography column can be used to estimate the molecular
weight of proteins. Performing gel filtration chromatography on a crude protein
preparation and then testing the resulting fractions for enzyme activity would
allow correlation of enzyme activity to a range of molecular weights. If the
molecular weight of the enzyme active fraction from a gel filtration
84
chromatography step is the same as the calculated molecular weight of purified
AADC1then existence of such an enzyme complex is not likely. If the two values
are different the existence of such an enzyme complex is more likely or AADC
may exist as a multimer that is not covalently linked by disulfide bridges.
The isoelectric point (pi) of AADC from P. raistrickii isolate H10BA2 was
determined by isoelectric focusing electrophoresis of a partially purified enzyme
preparation. The pi represents the pH at which the protein has a net neutral
charge. Results of this experiment indicate that the enzyme has a pi between
6.2 and 6.4. That there was only one peak of enzyme activity present within the
gel indicated that there was only one AADC enzyme present in this protein
preparation. There is at least a 0.4 pH unit difference between the enzyme’s pi
and optimum pH for enzyme activity, which is to be expected since a protein is
more likely to precipitate out of solution where the pH = pi, thereby removing the
enzyme activity (25). Table 4 showed that each AADC isolated from a different
source has a unique pi value. This indicates that these enzymes have
heterogeneous surface structures and therefore have unique amino acid
sequences to make such a variety of surface structures. Differences in amino
acid sequence can also be assumed by the difference in molecular weight of
these enzymes (see table 4).
The effect of the suicide inhibitors a-fluoromethyItyrosine and afluoromethyl (3,4-dihydroxyphenyl)alanine on AADC activity was determined (see
table 5). These suicide inhibitors did not have any effect on the enzyme activity.
85
This demonstrated that the fungal AADC is similar to the AADC from Sanguinaria
rhizomes, which also was not effected by these inhibitors. These inhibitors are
thought to covalently bind to the Pxy-P cofactor and enzyme (see figure 6).
These enzymes may not use Pxy-P as a cofactor, which would explain why these
enzymes were not affected by these inhibitors.
Some type of functional group is
still required for catalysis of this reaction and would most likely react with these
inhibitors. As demonstrated in chapter 1, there are decarboxylases that utilize a
pyruvyl moiety rather than Pxy-P to catalyze the reaction. Though pyruvyl has a
different chemical structure than Pxy-P, it performs the same function and may
also react with the suicide inhibitors. Steric hindrance might prevent these
inhibitors from entering the enzyme active site because of the presence of an ocfluoromethyl group instead of a proton (see figure 37). If the inhibitor cannot
approach the active site, it cannot inhibit the reaction.
An attempt to detect AADC activity in a different isolate of P. raistrickii was
made. P. raistrickii ATCC # 48678 was isolated from decomposing Eucalyptus
leaves in Australia. This isolate was chosen to determine if it also had AADC
activity because it was the most similar morphologically to the P. raistrickii
isolated from Taxus brevifolia. P. raistrickii ATCC # 48678 dids not have
detectable AADC activity in crude protein extracts for cultures incubated for 7
and 17 days. This fungus may produce AADC under different growth conditions,
however, but not in conditions in which it is produced by H10BA2. The crude
protein extracts of P. raistrickii isolated from Taxus brevifolia did contain enzyme
86
activity during these times (see figure 24). P. raistrickii ATCC # 48678 does not
produce taxol(85), but the P. raistrickii isolated from Taxus brevifolia does
produce taxol (18). Other differences in the production of secondary metabolites
Figure 37. Three dimensional comparison of a-fluoromethyltyrosine and
tyrosine.
Fluoromethyl
87
between these two fungi have not been investigated. This demonstrates that
AADC activity is not ubiquitous in the species P. raistrickii when different isolates
are grown under identical conditions. /VXDC activity may be correlated to
secondary metabolite production in specific strains.
88
CHAPTER 3
TRANSFORMATION OF Penicillium raistrickii
Introduction
This chapter presents the materials and methods used to develop a
transformation protocol for P. raistrickii isolate H10BA2, the results obtained from
these methods, and a discussion of the results. The plasmid used was pAN71
(61). This plasmid contains the Aspergillus nidulans gpd promoter region with
the Escherichia coli hph gene fused at the translation start codon. Downstream
of the hph gene is the termination region of the Aspergillus nidulans trpC gene.
This was constructed in pUC18. This construct results in constitutive expression
of the hygromycin resistance gene from E. coli.
Development of a transformation system was performed so that gene
manipulation experiments could be performed in the future. Gene disruption of
aromatic L-amino acid decarboxylase, by a homologous recombination event,
could be used to identify the biological function of this enzyme in isolate H10BA2.
Introduction of genes involved in taxol biosynthesis with promoters that allowed
89
increased production of the gene product could result in production of taxol at
higher levels. Introduction of a taxol pump, such as a P-glycoprotein, would
facilitate the transfer of taxol from the mycelium to the media.
Materials and Methods
Harvest of P. raistrickii Conidia
P. raistrickii conidia are very hydrophobic and difficult to maintain as a
suspension in water. Several solutions were tested to identify a solution that
would allow for easy manipulation of conidia in a dispersed suspension. Test
tube slants that contained mycological media (MMA; 10 g/l Soytone140 g/l
glucose, 15 g/l agar; MM did not contain agar) with sporulating P. raistrickii were
used as sources of conidia. Five ml of water, 1 % Tween 80, 1% Triton x 100 or
MM with 5% glycerol were added to a slant of P. raistrickii and vortexed. A
sample of the solution was analyzed microscopically for dispersion of conidia.
Sterile 1 % Tween 80 was used for subsequent harvesting of conidia. The
number of conidia per ml was determined with a hemacytometer.
Selection of Wild Type P. raistrickii with Hvaromvcin B
To identify what concentration of hygromycin B prevented the growth of P.
raistrickii conidia, a suspension of 1x104 and 1x10 6 conidia were spread over
90
MMA plates containing different concentrations of hygromycin. Hygromycin
concentrations tested included O1 100, 200,and 300, 400 and 600 p,g/ml.
Determination of Conditions for Germination of P. raistrickii Conidia
A suspension of P. raistrickii conidia was used to inoculate 25 ml of sterile
media in a 125 ml Erlenmeyer flask. Media tested included MM, MM + 2%
glycerol, and MM + 5% glycerol. Flasks were incubated at room temperature
with gentle shaking. Percent germination was determined semi-quantitatively by
microscopic examination throughout a 13 h incubation. A hemacytometer was
not used because cells formed aggregates that could not be analyzed in this
manner.
Formation of Protoplasts
Conditions for the formation of protoplasts from germinated conidia were
tested. Osmoticum solutions tested for protoplast formation included autoclaved
solutions of 10 mM Na^POvt, 10 mM C aC bat pH 5.8 containing either 0.6 M
KCI, 1.2 M KCI, 0.6 M sorbitol, or 1.2 M sorbitol. Germinated conidia (1x106
conidia per treatment) were harvested by centrifugation at 3000 x g for 1 min and
supernatant removed, concentrated by centrifugation at 5900 x g for 2 min. The
supernatant was removed to leave a final volume of 2 ml. One ml of 20 mg
Novozyme 234(BiosPacific) per ml of osmoticum was added to each treatment
91
and incubated at room temperature under still conditions. Protoplast formation
was determined by microscopic examination at hourly intervals.
Transformation Protocol
The method used for transformation of P. raistrickii was based on the
method developed for P. chrysogenum by Cantoral et. al. (86). Germination
media (150 ml MM + 5% glycerol in a 500 ml Erlenmeyer flask) was inoculated
with 2 x 10 7 conidia, and incubated at room temperature with gentle shaking for
at least 10 h for conidia to germinate. Germinated conidia were harvested by
centrifugation at 700 x g for 2 min in a swinging bucket rotor. Germinated conidia
were washed 2 times with 0.6 M Sorbitol, 50 mM NaH2PCU, 0.5 % Bovine serum
albumin at pH 5.8 (SPB) after supernatant was removed, 10 m l of SPB was
added, vortexed briefly, and centrifuged as before. The supernatant was
removed so that only 3 ml remained. One ml of 0.2 pm filter sterilized
Novozyme 234 (25 mg/ml SPB) was added to the washed conidia and incubated
at room temperature under still conditions. Formation of protoplasts was
determined microscopically. Protoplasts were divided into three 2 ml eppendorf
tubes, and centrifuged at 350 x g for 1 min. The supernatant was removed to
leave a final volume of approximately 250 pi. DNA was added to protoplasts at
0, 2 and 20 pg. Sixty pi of 50% polyethylene glycol 8000, 50 mM CaCI2, 10 mM
MBS, pH 5.8 (PCM) was added to each treatment and incubated on ice for 30
min. One ml of PCM was added to each treatment, mixed gently and incubated
92
at room temperature for 20 min. Seven hundred and fifty
of 0.6 M sorbitol, 50
mM CaCb, 10 mM IVIES, pH 5.8 was added to each treatment and mixed gently.
Aliquots of each treatment were spread onto plates of IVIIVIA + 200 jug/ml
hygromycin.
After incubation, colonies formed and were designated as putative
transformants. Putative transformants were transferred serially onto plates
containing MMA + 300 pg/ml hygromycin . To determine if the putative
transformants were stable, they were transferred to MID plates (73) to induce
sporulation under non-selective conditions. Conidia were isolated as described
previously and spread onto MMA + 100 p,g/ml hygromycin.
Results
Harvest of P. raistrickii conidia
Four solutions were tested for their ability to produce uniform suspensions
of P. raistrickii conidia. Sterile water and MM + 5% glycerol did not disperse the
conidia but maintained large aggregates of conidia. Sterile 1% Tween 80 and
1% Triton x 100 dispersed the conidia into a more uniform suspension. There
were strings of up to 3 conidia present in these solutions. It was decided to use
1% Tween 80 in subsequent experiments.
93
Selection of Wild Type P. raistrickii with Hygromvcin B
Results of spreading P. raistrickii conidia on hygromycin containing plates
are presented in table 6. The two colonies that arose on the 100 p.g/ml
hygromycin plates did not grow beyond 2 mm and did not produce colonies when
transferred to non-selective media.
Table 6. Selection of PeniciIIiLim raistrickii conidia on mycological agar plates
containing hygromycin. The rlumber of colonies that formed for each inoculation
density is reported after 7 and 14 days of incubation.
hygromycin concentration,
104 conidia per plate
10b conidia per plate
i-ig/ml
7days
14 days
7 days
14 days
0
>100
>100
>100
>100
100
0
0
0
2
200
0
0
0
0
300
0
0
0
0 .
Determination of Conditions for Germination of P. raistrickii conidia
Conidia of P raistrickii were inoculated into MM, MM + 2% glycerol, and
MM + 5% glycerol, and the time required for germination to occur is reported in
table 7. Germination was defined as production of a germ tube that was as long
as a conidium. As conidia germinated and the germ tubes lengthened,
aggregates of germinating conidia would form.
94
Table 7. Determine tion of germination of P. raistrickii conidia in mycological
media with different concentrations of glycerol added. The time required for a
percentage of conid ia to germinate are reported. ND=not determined
Time (h)
0% glycerol
2% glycerol
5% glycerol
2
0
ND
ND
4
0
ND
ND
9
>50 %
>75%
1%
10
ND
ND
1%
11
ND
ND
20%
12
ND
ND
50%
13
ND
ND
>50%
Formation of Protoplasts
Formation of protoplasts from germinated P. raistrickii conidia with different
osmotica were determined (see table 8).
Table 8. Formation of protoplasts from germinating conidia of P. raistrickii in
different osmotica. The progression of protoplast formation over time is reported
under each treatment.
Time (h)
0.6 M KCI
0.6 M Sorbitol
1.2 M Sorbitol
1.2 M KCI
No protoplasts
No protoplasts
1
No protoplasts
No protoplasts
2
No protoplasts
No protoplasts
No protoplasts
No protoplasts
3
Protoplasts
Protoplasts
Protoplasts
Protoplasts
forming
forming
forming
forming
Protoplasts
Cell debris
Protoplasts
Protoplasts
4
Cell debris
Protoplasts
5
Cell debris
Protoplasts
Transformation Protocol
After following the transformation protocol and placing protoplasts onto
selective media, colonies formed on plates from cells that had DNA added and
not added to them. Upon serial transfer of colonies onto MM + 300 ng/ml, there
95
was no growth from the colonies arising from controls lacking pAN71 DNA, while
colonies from treatments to which pAN71 DNA had been added continued to
grow. A persistent problem throughout the process was that the conidia and
protoplasts would form aggregates. This made it difficult to determine the
transformation efficiency, i.e. number of transformants/ p.g DNA. To test the
stability of the transformants, they were transferred to non-selective M1D media
to induce sporulation. Conidia were spread on a selective medium (MM + 100
jLig/ml hygromycin). Conidia from some of the putative transformants gave rise to
colonies on the selective media. No further analysis of putative transformants
was performed.
Discussion
In developing a transformation protocol for a fungus several preliminary
decisions were made, including what form of the fungus should be used, and
what vectors should be used. Conidia were chosen as the fungal state to
transform for several reasons. First conidia are haploid, uninucleate cells, while
mycelia can be multinucleate. With haploid, uninucleate cells there would be
fewer problems with selection of phenotypes and subsequent identification of
genotype later in the transformation process. Second, if the transformation
process were used to knock out a gene of interest by homologous recombination,
it can be accomplished more easily with such cells. Third, conidia were much
easier to manipulate and quantitate than mycelia when forming protoplasts.
96
Formation of protoplasts from conidia would be accomplished by treating
germinated conidia with cell wall removing enzymes. Experiments were
performed to identify conditions under which conidia germinate.
The plasmid used for transformation experiments was pAN71 (61). This
plasmid was chosen because it was the most commonly used plasmid for
transformation of Penicillium species(52, 54, 55). This construct allowed for the
selection of PeniciIHum transformants on hygromycin B-containing media. The
plasmid pAN7-1 has been used to transform P. roquefortii (52), P. paxilli (54),
and P. freii (55).
The conidia of P. raistrickii are very hydrophobic. A 1% (v/v) solution of
Tween 80 resulted in a uniform suspension of conidia. This solution was utilized
to harvest and quantitate conidia.
To make sure that wild type P. raistrickii was
not resistant to hygromycin B, different amounts of conidia were spread onto
mycological agar plates containing different concentrations of hygromycin B. The
data in table 6 showed that viable fungal colonies did not form when conidia were
spread onto mycological agar plates containing 200 jig hygromycin per ml. Two
small colonies did form on mycological agar plates containing 100 ^g hygromycin
per ml inoculated with 106 conidia. Those colonies did not grow after transfer to
non-selective media. These colonies might have expressed a transient
resistance to hygromycin B, but were not stable. This demonstrated that wildtype P. raistrickii can be selected against on plates of mycological medium
containing 200 p.g hygromycin B per ml.
97
Conidia were resistant to enzymes used for the formation of protoplasts
from mycelia. Protoplasts could be formed from germinated Conidia1 Different
conditions for the germination of conidia from P. raistrickii were tested (see table
7) . Five % glycerol in mycological media was used to germinate conidia because
this allowed for an overnight incubation. Glycerol was the only additive to
mycological medium that was tested. Tween 80, from the conidia solution,
constituted less than 0.1 % of the germination media solution.
Different conditions for the formation of protoplasts were tested (see table
8) . Stable protoplasts from germinating conidia of P. raistrickii were formed in
0.6 and 1.2 M sorbitol while protoplasts formed in 0.6 and 1.2 M potassium
chloride lysed (see table 8). Protoplasts were formed in 0.6 M sorbitol in
subsequent experiments. These conditions are similar to those used for P.
paxilli.
The transformation protocol, described in the materials and methods,
resulted in colonies that were resistant to hygromycin B at a concentration of 200
p,g per ml mycological medium. Growth of colonies on selective media is not a
definitive method to indicate the formation of stable transformed fungi. To
determine if stable transformants were obtained, putative transformants were
grown on non-selective media and the resulting conidia transferred back to
selective media. If the putative transformants had not been stable, then no
colonies would have grown on selective media. This was also a way to be
certain that colonies contained only transformed nuclei. It is possible that
98
germinating conidia used as the source for protoplasts contained more than one
nucleus per cell and transformation of just one nucleus in such a protoplast
would result in a heterokaryon. Only one nucleus is present in each conidium so
selecting for conidia containing transformed nuclei would segregate transformed
from non-transformed nuclei. Colonies did form after this process and indicated
that the putative transformants were stable and the colonies were homokaryons.
This still did not constitute definitive proof of transformation of P. raistrickii with
novel DNA. Southern blot analysis of transformant genomic DNA probed with
labeled pAN7-1 DNA would have been definitive proof that the transformants did
contain novel DNA. These experiments were not performed.
99
CHAPTER 4
INVESTIGATIONS OF TAXADIENE SYNTHASE ACTIVITY IN
PeniciIHum raistrickii ISOLATE H10BA2
Introduction
This chapter outlines the materials and methods used to investigate the
occurrence of taxa-4(5), 11 (12)-diene synthase activity in PeniciIHum raistrickii
isolate H10BA2. The results of this investigation are presented.
Materials and Methods
Fungal Culture
P. raistrickii isolate H10BA2 was utilized in these experiments. For the
production of taxadiene synthase, a final concentration of Ix IO 5 conidia/ml was
added to 200 ml M1S medium / 500 ml erlenmeyer flask. Cultures were
incubated at 25 °C at 200 rpm for days. Maximum taxol production occurred at
day 6, so cultures were harvested at day 5 to assay taxadiene synthase.
100
The assumption that the fungal enzyme would be very similar to the plant
enzyme was made. Identification of taxa-4(5), 11(12)-diene synthase activity
from the fungus was based on this assumption.
Chemicals
A litra n s geranylgeranylpyrophosphate, [1-3H] (15 Ci/mmole) was purchased
from American Radiolabeled Chemicals, Inc. All trans
geranylgeranylpyrophosphate was purchased from Sigma. Substrates were
combined to give a final specific activity of 85 Ci/mole. Addition of 7.5 pi of
substrate to 500 pi of enzyme assay solution gave a final concentration of 15 pM
all trans geranylgeranylpyrophosphate.
A taxa-4(5), 11(20)-diene, [20-13C]
standard was provided by Dr. Robert Williams at Colorado State University.
Buffers
The following buffers were used: wash buffer (0.1 mM
phenylmethylsulfonylfluoride (PMSF), 1 mM EDTA1pH 8.0), extraction buffer (20
mM magnesium chloride, 10% [v/v] glycerol, 1 mM dithiothreitol, 0.1 mM sodium
ascorbate, 30 mM (N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid]
[HEPES], pH 8.0), and assay buffer (1 mM magnesium dichloride, 10% [v/v]
glycerol, 1 mM dithiothreitol, 0.1 mM sodium ascorbate, 30 mM HEPES, pH 8.5.
Buffer C: 30 mM HEPES, pH 8.0). Wash buffer contained PMSF as an inhibitor
of serine proteases (87) and EDTA to chelate cations that may be required by
101
other proteases. Magnesium was added to extraction and assay buffers
because it is required by diterpene cyclases for enzyme activity (88). Glycerol,
dithiothreitol and sodium ascorbate were, added to help stabilize the enzyme.
Anion exchange (AE) buffers consisted of extraction buffer that varied in
concentration of potassium chloride. AE buffers 50, 100, 150 and 200 contained
50 mM, 100 mM, 150 mM and 200 mM potassium chloride, respectively.
Harvest of Protein
Mycelium was harvested on day five by filtering through cheesecloth.
Mycelium was soaked in 11of wash buffer and collected by filtration with
cheesecloth. The mycelium weighed 100 g. All subsequent steps were
performed at 4 °C. Mycelial cells were broken open by grinding the mycelium to
a fine powder in a ceramic mortar and pestle with addition of liquid nitrogen and
sand. Ground mycelium was placed into 350 ml of extraction buffer, to which 15
g of XAD-4 and 15 g of polyvinylpolypyrrolidone (PVPP) was added. XAD-4 and
PVPP were added to trap any compounds that could interfere with enzyme
activity. The slurry was centrifuged at 5,000 x g for 10 min to remove cell debris.
The resulting supernatant was centrifuged at 25,000 x g for 30 min. This crude
supernatant was utilized in the subsequent chromatography step.
102
Anion Exchange Chromatography
Whatman DE-52 anion exchange chromatography medium was used.
Twenty five g of DE-52 medium was prepared by soaking in 100 ml of 100 mM
potassium chloride solution. The DE-52 medium was poured into a
chromatography column and rinsed with 100 ml of buffer C. The crude
supernatant was applied to the DE-52 column. Five ml fractions were collected
from the column after the supernatant was loaded. The column was rinsed with
20 ml of buffer C, Protein was eluted by a stepped gradient of 25 ml of AE-50,
25 ml of AE-TOO150 ml of AE-150, and 25 ml of AE-200 buffer. Selected
fractions were assayed for enzyme activity, as well as crude supernatant.
Enzyme Assay
Assays for taxadiene synthase activity were performed by combining 200 pl
of protein solution with 300 pi of assay buffer and 7.5 pi substrate, to give a final
concentration of 15 pM geranylgeranylpyrophosphate. Substrate contained alltrans geranylgeranylpyrophosphate [1-3H] at a specific activity of 85 Ci/mole.
Enzyme assays were performed in sealed glass tubes rather than plastic,
because substrate would bind to plastic. Samples were incubated for 3 h at 31
°C in a water bath and extracted twice with 1 ml of hexane. Hexane extracts
were passed through individual silica columns that were prepared immediately
prior to use by filling a glass wool plugged Pasteur pipet to 40% volume with
silica gel and topped by a 3 mm thick layer of anhydrous magnesium sulfate.
103
The column was rinsed with 1 ml of hexane. The combined hexane effluent and
rinse was transferred to liquid scintillation vials containing 10 ml of a liquid
scintillation cocktail. The vials were counted with a liquid scintillation counter for
5 minutes.
Enzyme assays were extracted with hexane to separate the hydrophobic,
non-polar enzyme product from the aqueous assay solution. Hexane extracts
were passed through silica columns to separate the non-polar enzyme product
from any polar enzyme substrate or geranylgeraniol that would be co-extracted.
Geranylgeranylpyrophosphate and geranylgeraniol would bind to .the silica gel
and not be eluted by hexane. A layer of anhydrous magnesium sulfate was
included in the columns to trap any water that may be transferred with the
hexane extract.
First Attempt to Identify Enzyme Product
A culture, with a total volume of 2.4I, of H10BA2 was started, protein
harvested and anion exchange chromatography performed. AE-150 fractions
were used because that was the salt concentration at which the yew enzyme
eluted from the anion exchange column and because there was enzyme activity
in that fraction by liquid scintillation counting analysis. One half of the AE-150
fractions were concentrated from 20 ml to 2 ml utilizing an Amicon Centfiplus 30
concentrator. Enzyme assays were performed as described above, but the
enzyme assay would normally be gently extracted with 1 ml of hexane prior to
104
the addition of geranylgeranylpyrophosphate substrate. This extraction should
be done to remove any contaminating compounds that could cause a significant
background for GC-MS analysis making it impossible to identify a taxadiene
product, but was accidentally omitted. The organic extract was analyzed by GCMS by Al Koepp in Dr. Rodney Croteau’s laboratory at W ashington State
University (71).
Second Attempt to Identify Enzyme Product
A fungal culture was started by addition of 5x106 conidia per 2I Erlenmeyer
flask. Twelve 2I erlenmeyer flasks were inoculated and contained 500 ml of M1S
media per flask. Cultures were incubated as described above. Mycelium was
harvested as described above except the wash buffer was a solution of 10 mM
EDTA1 0.9% NaCI at pH 8.0. This and all subsequent steps were performed at 4
°C. Mycelium was ground with a mortar and pestle with addition of liquid
nitrogen. The powder was transferred to 600 ml of extraction buffer to which 60
g/l of XAD-2 and 60 g/l of PVPP was added. The slurry was centrifuged at
20,000 x g for 10 min. The supernatant was filtered through glass wool and
recentrifuged under the same conditions. Anion exchange chromatography was
performed with a column containing 100 ml of Bio - Rad Laboratories Macro Prep DEAE ion exchange support previously equilibrated with buffer C. The
supernatant was applied to the column and the column was washed with 60 ml of
buffer C. Protein was eluted with a stepped gradient of 60 ml AE-100, 105 ml
105
AE-150, and 210 ml of AE-200, with collection of 15 ml fractions. Enzyme
assays were performed on AE-150 fractions # 2 through 7 and AE-200 fractions
#1 through 3 as described above, except the silica columns were rinsed with 2 ml
of ethyl acetate after the hexane extract had been applied. Hexane and ethyl
acetate column rinses were counted separately by liquid scintillation counting as
described above. Columns were rinsed with ethyl acetate because previous
experiments showed that the hexane rinse contained a relatively low amount of
the proposed enzyme activity relative to the ethyl acetate rinse. It was assumed
that the enzyme product contained a hydroxyl group corresponding to a
taxadienol rather than geranylgeraniol, which would elute during an ethyl acetate
rinse.
Experiments to identify the enzyme product by GC/MS were performed on
the GC-MS system at Montana Tech described in chapter 2. The enzyme assay
consisted of 1.5 ml assay buffer, 1.0 ml of AE 150#7 fraction, and 20 pi of Sigma
geranylgeranylpyrophosphate. The assay solution was extracted gently 2 times
with 1 ml hexane prior to addition of the substrate. The assay was incubated for
6 h at 31 °C, and extracted 3 times with 1 ml hexane. Hexane extracts were
passed through a silica column followed by 2 -ml of ethyl acetate and dried under
a stream of argon and stored at -7 0 °C. The extract was analyzed by GC-MS at
Montana Tech. Gas chromatograph parameters were set with the injector at 270
°C, detector at 280 °C, initial oven temperature at 85 °C for 2 min with a ramp of
10 0CI min to 270 °C for 5 min, for a total run time of 25.5 min. The total ion
106
chromatogram generated by this analysis was analyzed for the presence of a
fragment ion of 272. A fragment ion of 272 corresponded to the molecular ion of
taxadiene, although at this time a taxadiene standard was not available.
Effect of pH on Enzyme Activity
Protein was prepared from a fresh culture of H10BA2 in the same manner
described above in Second Attempt to Identify Enzyme Product. Assay buffers
contained 1 mM magnesium chloride, 5 mM sodium ascorbate, 10% glycerol
(v/v), 5 mM dithiothreitol, and 30 mM buffer. Buffers used at 30 mM
concentration were ascorbate (AS) at pH 4.0, citrate (CT) at pH 4.5 and 5.0, 2[N-Morpholino]ethanesulfonic acid (ME) at pH 5.5, bis[2Hydroxyethyl]iminotris[hydroxymethyl]methane (BT) at pH 6.0, 3-[NMorpholinojpropanesulfonic acid (MP) at pH 6.5, tris (TS) at pH 7.0, 7.5, 8.0, and
9.0 and HEPES (HP) at pH 8.5. Enzyme assays were performed as described
above, but the volumes used were 200 pJ buffer, 50 pi protein solution, and 3 pi
substrate.
Identification of Enzyme Product
GC-MS analysis of enzyme reaction products was performed by Dr. Joe
Sears at Montana State University - Bozeman, Mass Spectrometry Facility
because this system was more sensitive than the system available at Montana
Tech. Gas chromatograph conditions were injector temperature at 260°Ci
107
column temperature begin at 50 °C, with a ramp of 50 °C/min to 100 °C then 5
0CZmin to 300 0C. A HP-5 column was used. Electron impact analysis was
performed.
Preparative enzyme assays were performed at pH 5.5 and 8.5 to determine
if the same product was being formed at each pH value. Protein was harvested
and prepared as described previously. The AE-150 active fraction was used as
protein source. Enzyme assays consisted of 800 pl buffer, 200 pi protein, and 10
pi of Sigma geranylgeranylpyrophosphate and boiled protein controls were made.
Five replicates of each pH treatment were made and incubated as previously
described. Each treatment was extracted 2 times with hexane. One silica
column was used for each pH treatment. The silica column was rinsed with 1 ml
of hexane, followed by 2 ml of ethyl acetate. The ethyl acetate was collected
separately in a glass round bottom flask and dried in vacuo. Samples were
purged with nitrogen gas and stored at - 4 °C until analyzed.
Results
First Attempt to Identify Enzyme Product
Putative taxadiene synthase activity was detected by liquid scintillation
counting analysis of enzyme assays (see figure 38). Background activity was at
154 DPM from the boiled 150 #5 control. Enzyme activity was present in
fractions 50 #3, 150 #6 and 8, and 200 #4.
108
Figure 38. Results from liquid scintillation counting analysis of enzyme assays of
anion exchange fractions from the first attempt to identify enzyme product.
Anion Exchange Fraction, AE fraction number
5
Because the enzyme assay performed for GC-MS was not extracted with
hexane prior to the addition of substrate no taxadiene or any other product could
be identified at that time by GC-MS analysis.
Second Attempt to Identify Enzyme Product
DPM contained in the hexane and ethyl acetate rinses of the silica columns
used for enzyme assays of an anion exchange column are presented in figures
39 and 40, respectively. Figure 39 shows that there was potential enzyme
activity from the hexane rinses of fractions 150-3, 5, 6, and 7 when compared to
the boiled control. This enzyme activity may be attributed to a taxadiene
synthase. Figure 40 shows that there is significant enzyme activity from the ethyl
109
fractions 150-5, 6, and 7, and 200- 1, 2, and 3 when compared to the boiled
control. This enzyme activity may be attributed to a taxadienol synthase or
phosphatase.
Figure 39. DPM from the hexane rinse of the silica columns from enzyme assays
of selected fractions from an anion exchange column described in second
attempt to identify enzyme product.
250
200
Ol
Oi
M
CO
o
O
CD
O
I
ro
Ol
O
Ol
O
Oi
O
O
O
ro
O
O
Anion Exchange fractions-AE fraction number
ro
O
O
CD
O
110
Figure 40. DPM from the ethyl acetate rinse of the silica columns from enzyme
assays of selected fractions from an anion exchange column described in second
attempt to identify enzyme product.
450000
400000
350000
300000
S 250000
Q 200000
150000
100000
50000
0
c n c n c n c n o i
o
o
o
o
o
o
M
C
J
-
t
^
c
n
c
n
^
c n o o o
o
o
o
J
-
L
M
O
J
Anion Exchange fractions-AE fraction number
o
Ol
o
GC-MS analysis was performed to identify the enzyme product of the
processed enzyme assay and the results are presented in figures 41 and 42.
Ions that would indicate the presence of a hydroxylated taxadiene have a mass
of 272, 257, and 229, as indicated by the fragmentation pattern of verticillol (see
figure 43). Figure 43 contains a reproduction of the fragmentation pattern of
verticillol (89) as an example of a hydroxylated cyclized diterpene. The total ion
count spectrum was analyzed for the presence of these ions, and two peaks that
eluted between 16.7 to 16.74 and 16.9 to 16.92 minutes contained at least two of
these ions. However, the occurrence of the 272 ion fragments in figures 41 and
111
Figure 41. GC-MS analysis of products from an enzyme assay. The top graph is
the total ion count of the peaks eluting between 16.50 and 17.00 min. The
bottom graph is the fragmentation pattern of the peak between 16.702 and
16.736 min.
m ndance
tIC :
C7150D .D
100000 -
80000
60000 .
16.9-16.92 ,
rS I
40000
16.7-16.74
20000
16.60
rim e --> 1 6 .5 0
A v e ra g e
1000 J
600
400 _
200
m /z-->
160
iio
o±
16.70
1 6.702
to
16.80
16.736
m in .:
16.90
C 7150D .D
(+ ,*)
112
Figure 42. Fragmentation pattern of a peak between 16.901 and 16.921 min
from the analysis in figure 41.
abundance
2 8 0 0 -I
2600 :
2400 .
2200
-
2000
:
1800 :
1600 :
1400 :
1200 :
iooo:
aoo:
eoo:
4oo:
200:
I
I
I
I
I
T
160
l
I
I
I
120
>
'
I
I
*
140
2 io
'
24o ' ' 266 '
113
Figure 43. Structure of verticillol and it’s mass spectral fragmentation pattern
taken from the work of Bengt Karlsson et. al. (89).
O
20
40
60
80
100 120 140 160 180 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 00
m/z, fragm ent mass
42 indicate the presence of a diterpenoid compound. The high abundance of
229 ions, in figures 41 and 42, relative to the smaller molecular weight ion
fragments is indicative of a cyclized diterpene product rather than the linear
114
diterpene geranylgeraniol. The fragmentation patterns of these peaks were not
identical to the pattern of taxadiene or verticillol.
Effect of pH on Enzyme Activity
The effect of pH on enzyme activity measured by liquid scintillation counting
analysis of the combined hexane and ethyl acetate rinses of the silica columns
used for each pH treatment is presented in figure 44. Figure 44 shows that
enzyme activity peaks at pH 5.5 and is present to pH 8.5.
Figure 44. Effect of pH on enzyme activity. Enzyme was from the AE-150 active
fraction of an anion exchange column. Activity is reported as the DPM present in
the combined hexane and ethyl acetate rinse of a silica column from each
treatment.
70000
60000
50000
:> 40000
CL
Q
30000
20000
-
10000
I
0
3.5
4
4.5
5
5.5
6
6.5
pH
7
7.5
8
8.5
9
9.5
115
Identification of Enzyme Product
Figures 45 to 54 presented the results of GC-MS analysis performed at the
Montana State University - Bozeman Mass Spectrometry Facility of standard
compounds and enzyme extracts as described in the materials and methods.
Figure 45 shows the total ion count analysis of a taxadiene [20-13C] standard.
This figure demonstrated that the taxadiene standard eluted as peak 1531 and
that there were other compounds present in the standard. The hydroxyl group
present in a taxadienol would cause it to elute at a later time. The fragmentation
pattern of a shoulder of the taxadiene peak is presented in figure 46. Since the
standard contained 1 13C molecule in the structure it is necessary to subtract one
from most of the peaks presented in figure 46 to get the naturally occurring ion
mass.
After comparing the taxadiene standard (figure 46) and verticillol (figure
43) diagnostic ion fragments that would indicate a taxadiene or taxadienol would
be 290 (molecular ion), 272, and 257. The relatively high abundance values for
ions 272 and 257 to the lower molecular weight ions are indicative of a cyclized
diterpenoid product.
Figure 47 shows the total ion count analysis of a geranylgeraniol standard.
This figure demonstrated that the geranylgeraniol standard eluted as peak 1909
and that there were other compounds present in the standard. This standard
was analyzed because the most likely enzyme activity occurring in the AE-150
fractions could be attributed to phosphatase activity. Phosphatase would result
in the production of geranylgeraniol. The fragmentation pattern of the
Figure 45. The total ion count analysis of a taxa-4(5), 11(20)-diene [20-13C] standard (peak 1531).
e g : 9 - F E B - 1 9 9 6 0 9 : 2 1 : 3 9 GC E I +
P i l e : 9FEB96B # 1 -3 1 3 7
T IC (+R F)
E x p : GC-MS:I
S a m p le T e x t : m a - 1 7 - 2 l b c d e F i l e T e x t : t a x a d i e n e c l 3
IO O i
1531
M agnet
70S
7 .3 E 7
7 .0 E 7
90 J
L 6 .6 E 7
85J
L 6 .2 E 7
80j
L 5 .9E 7
IS l
L 5 .5E 7
70 J
L 5 .1E 7
65 j
L4 . 8E7
60J
4 .4 E 7
55j
4 .0 E 7
50 j
3 .7 E 7
45 j
L 3 .3E 7
40J
L.2.9E 7
35 J
L 2 .6E 7
30J
2 .2 E 7
25 J
L1.8E7
2131
2338
20 j
15
10:
1 .5 E 7
1908
2533
1789
L 1 .1 E 7
L 7 .3 E 6
5j
L 3 .7E 6
0
O.OEO
Scan
T im e
116
95 j
Figure 46. The fragmentation pattern of a shoulder of the taxa- 4(5), 11 (20)-diene [20-13C] standard, shown
in figure 44. Subtract 1 from the masses shown to get the fragment mass of the naturally occurring
taxadiene.
F i l e : 9FEB96B I d e n t : 1 5 4 6 _ 1 5 b 2 - 1 5 9 0 _ l 6 0 4 M e r “ Se T ^D T2 5 A c q : 9 - F E B - l ? S F ~ 0 y : 2 1 : 3S) + 2 6 : 1 2 C a l: 9 F E B 9 6 A »
70S E I + M a g n e t BpM :1 2 3 B p I : 7 3 8 5 7 T I C : 1 0 5 2 5 4 4 F l a g s : H A L L
S a m p le T e x t : m a - 1 7 - 2 l b c d e F i l e T e x t : t a x a d i e n e c l 3
100 !
_ 7 . 4E4
L 7 . 0E4
L6.6E 4
L6.3E 4
L5.9E 4
L 5 . 5E4
L5.2E 4
l4
. 8E4
-4.1 E 4
l 3.3E 4
L3 . 0E4
i . 2 . 6E4
L2.2E 4
L i . 8E4
L i . 5E4
L i . 1E4
L 7 .4 E 3
L 3 .7 E 3
0.0E 0
117
L4.4E 4
Figure 47. The total ion count analysis of a geranylgeraniol standard (peak 1909).
F i l e : 22MAY96E # 1 - 2 7 6 6 A c q :2 2 - M A Y - 1 9 9 6 1 3 : 1 7 : 5 5
T I C (+H P )
E x p : N ID E N S
S a m p le T e x t :
F i l e T e x t : S t a n d a r d faL nO tf
1 0 0 %,
GC E I +
M agnet
70S
1909
r_2.8E7
1 2 .7 E 7
1 2 .6 E 7
L 2 .4 E 7
1 2 .3 E 7
L 2 .1 E 7
L 2 .0 E 7
L i . 8E7
L 1 .6 E 7
L i . 4E7
L I . 3E7
L i . 1E7
L 9 .9 B 6
-5 .7 E 6
4 .3 E 6
1000
11:11
1 8 :2 1
1500
2 5 :3 1
2000
3 2 :4 1
3 9 :5 2
3000
4 7 :0 2
Scan
T im e
118
L i . 7E7
119
geranylgeraniol peak in figure 47 is presented in figure 48. This fragmentation
pattern, of relatively low abundance of high mass ions, 290, to the low mass ions,
69 to 136, is characteristic of a linear diterpenoid compound. The diagnostic ions
290, 272 and 257 are also present in this compound in very low abundance.
Figures 49 to 51 show the analysis of enzyme product from enzyme assays
performed at pH 5.5. Figure 49 shows the TIC of the ethyl acetate rinse from the
silica column as plot A, a single ion scan for ion fragments (SIS) with a molecular
weight of 272 as plot B, and the SIS for 257 ions as plot C. Figure 49 plot A
demonstrated that there are several compounds present in the ethyl acetate
rinse of the silica column. No compound that would correspond to taxadiene was
detected in the 1531 region. A compound was present in the 1905
geranylgeraniol region. Plot B demonstrated that peak 1906 was the only peak
with an appreciable amount of the diagnostic ion 272. Plot C demonstrated that
the peak before peak 1906 was the only peak with an appreciable amount of the
diagnostic ion 257. Figure 50 shows the fragmentation pattern of the peak
centered at 1906 and demonstrated that this compound was geranylgeraniol
since it had the same fragmentation pattern and eluted at the same time as the
geranylgeraniol standard (see figure 47 and 48). Figure 51 contained the TIC for
the ethyl acetate rinse of a silica column from the boiled enzyme assay controls
performed at pH 5.5 as plot A and a SIS for 272 ions as plot B. Figure 51
demonstrated that there was no peak present at 1906 (plot A) and that there was
no peak at 1906 when scanning for the diagnostic 272 ion (plot B). This analysis
Figure 48.
The fragmentation pattern of the geranylgeraniol standard, shown in figure 46.
F ile :2 2 M A Y 9 6 E I d e n t : 1 9 0 6 _ iy iO - 1 8 8 7 _ 1 8 9 4 M e r D e f 0 .2 5 A c q : 2 2 -M A Y -1996
70S E I+ M a g n e t B pM :6 9 B p l: 5 0 3 8 6 6 0 T IC :2 2 3 5 2 4 4 4 F la g s r H A L L
S a m p le T e x t :
F ile T e x trS ta n d a rd
IO O i
1 3 :1 7 :5 5
+ 3 1 :2 2
C a l:2 2 M A Y 9 »
r_5 .0 E 6
L 4 .8 E 6
L 4 .5 E 6
L 4 .3 E 6
L 4 .0 E 6
L 3 .8 E 6
L 3 . 5E6
L 3 .3 E 6
L 3 .0 E 6
L 2 .5 E 6
L 2 .3 E 6
L 2 . 0E6
L i . 8E6
L i . 5E6
L i . 3E6
- I . 6E5
L5.0E5
i 2 . 5E5
0 . OEO
346 ' 3^6 ' 340 ' 366 ' 366 ' 460
120
L 2 .8 E 6
Figure 49. GC-MS analysis of the ethyl acetate rinse of a silica column from an AE-150 enzyme assay
performed at pH 5.5. Plot A: total ion count analysis. Plot B: Ion 272 single ion scan of plot A. Plot C: Ion
257 single ion scan of plot A.
F i l e : 22MAY96B # l - i 2 7 6 A c q :2 2 -M A Y -1 9 9 6 0 9 : 5 2 : 3 6
T IC (+R F)
E x p : N ID E N S
S a m p le T e x t :
F i l e T e x t : b r c 3 6 - 1 5d
p h = 5 .5
IOO t
GC E I +
M agnet
70S
5 .7 E 7
8 0 j
2315
60J
L 4 . 6E7
1271
L 3 .4 E 7
4 OJ
L 2 .3 E 7
20j
1905
986
OJ
-F -
2128
I .
i
2000
500
1000
1500
1 1 :1 1
1 8 :2 1
2 5 :3 1
F i l e : 22MAY96B # 1 - 3 2 7 6 A c q :2 2 -M A Y -1 9 9 6 0 9 : 5 2 : 3 6
2 7 1 . 9 _ 2 7 2 .3 (+R F) M e r D e f 0 . 2 5
E x p : N ID E N S
S a m p le T e x t :
F ile T e x t:b rc 3 6 -1 5 d
p h = 5 .5
IOOt
3 2 :4 1
E I+ M a g n e t
L i . 1E7
2587
2500
3 9 :5 2
-LO .O E O
Scan
T im e
3000
4 7 :0 2
70S
1906
.2 .6 E 4
80J
L 2 .1 E 4
60J
4 OJ
L i . 5E4
.1 .0 B 4
1556
500
1000
1500
1 1 :1 1
1 8 :2 1
2 5 :3 1
F i l e : 22MAY96B # 1 -3 2 7 6 A c q : 2 2 -M A Y -1 9 9 6 0 9 : 5 2 : 3 6
2 5 6 . 9 _ 2 5 7 .3 (+RF) M e r D e f 0 . 2 5
E x p : N ID E N S
S a m p le T e x t :
F ile T e x t:b rc 3 6 -1 5 d
p h = 5 .5
100%
3156
2160
-4+
- T - u T -
2000
3 2 :4 1
GC E I + M a g n e t
2500
3 9 :5 2
3000
4 7 :0 2
.5 .1 E 3
. 0 . 0E0
Scan
T im e
70S
1896
. 3 . 3E5
80J
L 2 . 6E5
6Oj
J 2 .0 E 5
4 Oj
L i . 3E5
2 Oj
_ 6 . 6E4
0.
500
1000
11:11
1 8 :2 1
1500
2 5 :3 1
2000
3 2 :4 1
2500
3 9 :5 2
3000
4 7 :0 2
_ 0 . 0E0
Scan
T im e
Figure 50. The fragmentation pattern of the 1906 peak, shown in figure 48.
F ile :2 2 M A Y 9 6 B I d e n t : 1 9 0 2 _ 1 9 0 8 -1 8 2 0 _ 1 8 4 9 M e r D e t 0 .2 5 A c q : 2 2 -M A Y -I 996
7 0 S E I + M a g n e t B p M :69 B p I : 1 2 3 7 1 1 8 T I C : 4 8 2 4 3 2 7 F la g s r H A L L
S a m p le T e x t :
F ile T e x t:b rc 3 6 -1 5 d
p h = 5 .5
0 9 :b 2 :3 6
+ 3 1 :2 0
C a l : 22 mAy 9»
100%,
L i . 2E6
L i . 1E6
L i . 1E6
L 9 . 9E5
L9.3E 5
L8.7E 5
L8.0E 5
L6.8E 5
L6.2E 5
L 5 . 6E5
l
4.9 E 5
L4.3E 5
L3.7E 5
l
3 .1 E 5
L2.5E 5
L i . 9E5
L1.2E 5
L6.2E 4
0.0E 0
122
L7.4E 5
Figure 51. GC-MS analysis of the ethyl acetate rinse of a silica column from a boiled AE-150 enzyme assay
control performed at pH 5.5. Plot A: total ion count analysis. Plot B: Ion 272 single ion scan of plot A.
F i l e : 31MAY96A # 1 -3 4 9 3 A c q :3 1 - M A Y - 1 9 9 6
T IC (+R F)
E x p : N ID E N S
S a m p le T e x t :
b la n k F i l e T e x trN ie d e n s
100%,
0 8 :4 1 :2 8
b o il
GC E I +
b rc3 6
M agnet
70S
5 .5
2446
1 .4 E 7
90,
1 .2 E 7
8 0_
: 1 .1 E 7
70-
4 0_
L9 .7 E 6
L8 .3 E 6
L6 .9 E 6
L5 .5 E 6
30-
4 .1 E 6
60_
5 0_
20_
2 .8 E 6
1322
1 .4 E 6
V
0
500
1000
1500
2000
F i l e : 3 1 M A Y 9 6 A # 1 - 3 4 9 3 A c q : 3 1 - M A Y - 1 9 9 6 0 8 : 4 1 : 2 8 GC E I + M a g n e t
2 7 1 . 9 _ 2 7 2 . 4 (+R F ) M e r D e f 0 . 2 5
E x p rN ID E N S
S a m p le T e x t :
b la n k F i l e T e x trN ie d e n s
b o i l b rc 3 6 5 .5
.
2500
70S
3000
: 0.0E0
Scan
1 00 %
.2 .2 E 2
90j
. 2 . 0E2
80 j
. 1 . 8E2
7 OJ
_ 1 .5E 2
6 OJ
. 1 . 3E2
50j
.1 .1 E 2
40 J
.8 .8 E 1
3 OJ
.6 .6 E 1
2 OJ
. 4 . 4E1
IOJ
2 .2 E 1
OJ.
560
1000
1500
2000
'
I
2500
r
3000
0 .0 E 0
Scan
123
10
124
demonstrated that the enzyme activity present in AE-150, assayed at pH 5.5,
resulted in the production of geranylgeraniol and not taxadiene or a taxadienol.
This enzyme activity was most likely from a general phosphatase.
Figures 52 to 54 show the analysis of enzyme product from enzyme assays
performed at pH 8.5. Figure 52 shows the TIC of an ethyl acetate rinse of the
silica column as plot A, and a SIS for 272 ions as plot B. The fragmentation
pattern for peak 1906 in figure 52 is presented in figure 53. Figure 54 contained
the TIC for the ethyl acetate rinse of a silica column from a boiled enzyme assay
control as plot A, with the corresponding SIS for 272 ions as plot B. Figure 52
plot A demonstrated that there were several compounds present in the ethyl
acetate rinse of the silica column. No compound that would correspond to
taxadiene was detected in the 1531 region. A compound was present in the
1905 geranylgeraniol region, which was the shoulder of the 1896 peak. Plot B
demonstrated that peak 1906 was the only peak with an appreciable amount of
the diagnostic ion 272 since this peak was 3 times above background. The
fragmentation pattern of the peak centered at 1906 (see figure 53) demonstrated
that this compound was geranylgeraniol since it had the same fragmentation
pattern and eluted at the same time as the geranylgeraniol standard (see figure
46 and 47). The corresponding boiled enzyme control analysis in figure 54
demonstrated that there was no peak present at 1906 (plot A) and that there was
no peak at 1906 when scanning for the diagnostic 272 ion (plot B). This analysis
demonstrated that the enzyme activity present in AE-150, assayed at pH 8.5,
Figure 52. GC-MS analysis of the ethyl acetate rinse of a silica column from an AE-150 enzyme assay
performed at pH 8.5. Plot A: total ion count analysis. Plot B: Ion 272 single ion scan of plot A.
F i l e : 22MAY96C # 1 - 3 2 7 7 A c q :2 2 - M A Y ^ 1 9 9 6 1 0 : 4 7 : 5 6
T IC (+R F)
E xp :W ID E N S
S a m p le T e x t :
F i l e T e x t : b r c 3 6 - 15d
p h = 8 .5
GC E I +
M agnet
70S
100 %
,5 .7 E 7
9 03
L 5 .1 E 7
80_!
L 4 .6E 7
2314
70 j
L 4 .0 E 7
60J
1272
L 3 .4 E 7
50 j
1 2 .9E 7
40 j
L 2 .3 E 7
30J
-1 .7 E 7
20J
L 1 .1E 7
987
jL
500
1000
1500
1 1 :1 0
1 8 :2 0
2 5 :3 1
F i l e : 22MAY96C # 1 - 3 2 7 7 A c q : 2 2 - M A Y - 1 9 9 6 1 0 : 4 7 : 5 6
2 7 1 . 9 _ 2 7 2 .4 (+R F) M e r D e f 0 . 2 5
E xp :W ID E N S
S a m p le T e x t :
F ile T e x t:b rc 3 6 -1 5 d
p h = 8 .5
1 0 0 %,
1896
L 5 . 7E6
2129
_JL
2000
3 2 :4 1
GC E I + M a g n e t
1905
2500
3 9 :5 1
70S
3000
4 7 :0 1
L O . OEO
Scan
T im e
125
10J
oJL
Figure 53. The fragmentation pattern of the 1906 peak, shown in figure 51.
F i l e : 22MAY96C I d e n t : 1 9 0 2 _ 1 9 0 8 - 1 8 1 8 _ 1 8 3 2 M e r
7 0 S EX+ M a g n e t B p M :6 9 B p I : 1 2 7 1 2 0 T I C : 5 2 0 5 3 8
S a m p le T e x t :
F ile T e x t:b rc 3 6 -1 5 d
p h = 8 .5
100%,
69
D e f 0 .2 b A c q :2 2 -M A Y -1 9 9 6
F la g s :H A L L
1 0 :4 7 :5 6
+ 3 1 :1 9
C a l:2 2 M A Y 9 »
_ 1 . 3E5
L i . 2E5
L i . 1E5
L i . 1E5
L i . 0E5
l
9 .5 E 4
L8.9E 4
L 8 .3 E 4
L7.0E 4
126
L7.6E 4
L6.4E 4
L5.7E4
L5.1E4
L4.4E4
L3.8E4
L3.2E4
2 .5 E 4
L i . 9E4
-1 .3 E 4
L6.4E3
0 .0 E 0
—
Figure 54. GC-MS analysis of the ethyl acetate rinse of a silica column from a boiled AE-150 enzyme assay
control performed at pH 8.5. Plot A: total ion count analysis. Plot B: Ion 272 single ion scan of plot A.
F i l e : 31MAY96B # 1 - 3 4 9 2 A c q : i l - M A Y - 1 9 9 6
T I C (+ R F )
E x p : NIDEN S
S a m p le T e x t :
b la n k F i l e T e x t : N ie d e n s
100%
0 9 :3 6 :3 4
GC E I +
b o i l b rc 3 6
M a g n e t 70S
8 .5
2449
2 .3 E 7
901
L2 .0E 7
801
L1 .8E 7
701
501
L i . 6E7
L i . 4E7
L i . 1E7
401
L9 .0E 6
301
L6 .8E 6
201
L4 .5E 6
601
A
2246
JlLJL
L2 .3E 6
2738
3000
4 4 :2 2
500
1000
1500
2000
2500
1 0 :4 4
1 7 :2 8
2 4 :1 1
3 0 :5 5
3 7 :3 8
F i l e : 31MAY96B # 1 - 3 4 9 2 A c q : 3 1 - M A Y - 1 9 9 6 0 9 : 3 6 : 3 4 GC E I + M a g n e t 7 0 S
2 7 1 . 9 _ 2 7 2 . 4 (+R F ) M e r D e f 0 . 2 5
E x p : NIDEN S
S a m p le T e x t :
b la n k F i l e T e x t : N ie d e n s
b o i l b rc 3 6 8 .5
100%
. 0 . 0E0
Scan
T im e
4 .0 E 2
2740
L3 .6E 2
801
L3 .2E 2
70J
|_2. 8E2
60 j
L2.4E 2
50 j
L2.0E 2
401
L i . 6E2
L1.2E 2
301
191:!
201
101
01
L 7 .9E 1
L 4 .0E 1
' sio '
1 0 :4 4
_ L o . 0E 0
1 0 00
1 7 :2 8
1500
2 4 :1 1
2000
3 0 :5 5
2500
3 7 :3 8
3000
4 4 :22
Scan
T im e
127
19?7
1323
128
resulted in the production of geranylgeraniol and not taxadiene or a taxadienol.
This enzyme activity was most likely from a general phosphatase.
In comparing the analysis of enzyme product made at pH 5.5 and 8.5 it
should be noted that the 1905 peak in figure 49, plot A had an abundance of
almost 1.1x1071 but the corresponding peak in figure 52, plot A was barely
detectable. This reflected the relatively high enzyme activity present at pH 5.5 to
that present at pH 8.5 in figure 44.
Discussion
The assumption that the fungal enzyme would be very similar to the plant
enzyme was made. Identification of taxa-4(5), 11 (12)-diene synthase activity
from the fungus was based on this assumption. The same general protocol used
to isolate taxa-4(5), 11 (12)-diene synthase from the yew tree was followed
utilizing P. raistrickii isolate H10BA2 as the source in the first attempt to identify
the enzyme product. Figure 38 demonstrated that there was a potential for
enzyme activity in different fractions from the anion exchange chromatography
purification step. There was no enzyme activity present in the Crude protein
supernatant by liquid scintillation counting analysis. This was a typical result
found when using the yew tree as a source. This was explained by the enzyme
being very dilute in the crude supernatant, and that there would be competing
reactions, for enzyme substrate, such as phosphatases. Phosphatase activity
would not be detected in this assay because the product, geranylgeraniol, would
129
stick to the silica column and not be eluted during hexane rinses.
Geranylgeraniol would elute from the silica column if a more polar solvent such
as ethyl acetate were used. Potential enzyme activity was detected in the AE
fractions 50 #3, 150 #6 and 8, and 200 #4 (see figure 38). This activity was at
least 2 to 3 times above the boiled control value. This activity was much lower
than that found with enzyme isolated from the yew tree, which would be in the
tens of thousands of DPM. AE 150 fractions were used to identify enzyme
activity by GC-MS analysis of enzyme product because that was the salt
concentration at which the yew enzyme eluted. Due to an error in the procedure
enzyme activity was not identifiable by GC-MS analysis. Considering the low
enzyme activity (see figure 38) found in the fungal extract, relative to the known
enzyme activity from the yew tree, the possibility of isolating the enzyme was not
encouraging.
A second attempt to identify the enzyme product was made. Potential
enzyme activity was detected in the hexane and ethyl acetate rinses of the silica
columns from the corresponding enzyme assays (see figures 39 and 40). The
hexane rinses continued to show very little activity, with ranges between 100 and
200 DPM (see figure 39). This demonstrated that the potential taxadiene
enzyme product was probably not being formed. It was postulated that the
enzyme product could be hydroxylated (taxadienol) and be similar in structure to
the known compound verticillol (see figure 43)(89). This type of product would
130
elute from the silica column with more polar solvents such as ethyl acetate. The
ethyl acetate rinses showed very high potential enzyme activity (see figure 40).
Characterization of the enzyme product by GC-MS analysis at Montana
Tech was attempted (see figure 41 and 42). The fragmentation patterns shown
in figures 41 and 42 are indicative of a cyclized diterpene product rather than the
linear diterpene geranylgeraniol. The fragmentation patterns of these peaks
were not identical to the pattern of taxadiene or verticillol (see figures 46 and 43,
respectively). Unfortunately, these fragmentation patterns were not identified in
subsequent experiments. This gas chromatograph had been used for a variety
of analyses of samples of unknown origin and even though attempts were made
to clean the column, the quality of the column was suspect. For this reason
subsequent GC-MS analysis was performed at the Mass Spectrometry Facility at
Montana State University-Bozeman, to identify the enzyme product. This
equipment was also much more sensitive than that available at Montana Tech.
W ork was continued on the AE-150 fraction because of these preliminary results.
The effect of pH on the enzyme activity of the AE-150 fraction was
determined (see figure 44). Enzyme activity was present from pH 5.5 to 8.5.
This type of peak could be explained by the presence of one enzyme with a
broad range of activity or by the presence of several enzyme activities with
different pH optima being detected. As previously stated, it is not possible to
identify the structure of a compound by liquid scintillation counting analysis, but
that it is a good quantitative method once an assay system has been shown to
131
be specific for a compound. Since enzyme activity was present at both pH 5.5
and 8.5 the enzyme products produced in these two treatments were identified
by GC-MS analysis.
Figures 45 to 54 presented the results of GC-MS analysis of standard
compounds and enzyme extracts as described in the materials and methods.
This analysis demonstrated that the enzyme activity present in the AE-150
fractions, assayed at pH 5.5 and 8.5, resulted in the production of
geranylgeraniol and not taxadiene or a taxadienol. This enzyme activity was
most likely from a general phosphatase.
These experiments to identify taxa-4(5), 11(12)-diene or taxadienol
synthase activity in the taxol producing fungus P. raistrickii isolate H10BA2,
demonstrated that trying to follow the protocols developed for identification of
taxa-4(5), 11 (12)-diene synthase from the yew tree, resulted in the identification
of a phosphatase. This phosphatase produced geranylgeraniol from the
precursor geranylgeranylpyrophosphate (see figure 16).
132
CHAPTER 5
DISCUSSION
In this chapter I will discuss the work presented in chapters 2, 3, and 4 as
they related to each other. I will also place this work in context of what has been
described in the literature, and how this research can be further developed and
applied in the future.
All of this work revolved around the ability of the fungus P. raistrickii isolate
H10BA2 to produce secondary metabolites that have biological activity. R
raistrickii isolate H10BA2 produced the anti-cancer compound taxol and as such
has potential industrial application as a producer of taxol. Taxol is presently
utilized in the treatment of breast and ovarian cancer in women (18).
There has been a concern about there being an 'adequate supply of taxol for
cancer treatment. The problem was that the commercial source, Taxus brevifolia
or the yew tree, is a slow growing tree that occurs in limited numbers. It was
projected that the demand for taxol would far surpass the supply (90). It now
appears that the supply crisis has been resolved by utilizing other Taxus species
as sources of precursors for semi-synthesis of taxol. However, finding alternative
133
sources of taxol was, and still is, of interest. One idea was to find an endophytic
fungus from the commercial source of taxol, Taxus brevifolia, which would also
be a producer of taxol. A fungus would be amenable to large-scale fermentation
for production of taxol, thereby resolving the taxol supply crisis. This approach
was based on examples in the literature of fungi that produced compounds
thought to be only produced by plants. The bakanae (“foolish seedling”) disease
of rice is one example (43). In this disease, the fungus Gibberella fujikuroi infects
rice seedlings, causing them to be taller, chlorotic and sterile. This fungus
produces the plant hormones known as gibberellins, which cause the disease
symptoms. It could be considered that gibberellins should only be produced by
higher plants and not by a fungus, because these compounds serve a basic
physiological role in plant development which is not evident in fungi. Gibberella
fujikuroi, however, does produce the gibberellins, which cause disease
symptoms in plants resulting in a beneficial environment for the growth of the
fungus. This example of a fungus producing a “plant” compound for use by the
fungus led to the identification of several endophytic fungi from Taxus brevifolia
that produced taxol (18). P. raistrickii isolate H10BA2 was discovered in this
search.
It was reported later that the biological function of taxol in Taxus brevifolia
might be as part of the plant’s defense against some fungi. It has been shown
that taxol inhibits the growth of some plant pathogens including Phytophthora
and Pythium species (Oomycetes) and Rhizoctonia solani (Basidiomycetes)
134
(91,92). It might be advantageous for an endophytic fungus of Taxus brevifolia to
produce or at least metabolize taxol so that it could be resistant to this toxic
compound.
P. raistrickii isolate H10BA2, like the other taxol producing fungi isolated
and studied to date, produced taxol in the ng to pg/l range. To be commercially
applicable, a fungus would need to produce taxol in the mg/I range. One
approach to overcome the problem of fungi producing such small quantities of
taxol would be to study the enzymatic steps in the biosynthesis of taxol. Once an
enzyme had be,en isolated and characterized, the DNA sequence coding for that
enzyme could be cloned. Once the enzyme sequence was cloned, it could be
manipulated with the available DNA technology to produce industrial quantities of
taxol. P. raistrickii isolate H10BA2 was chosen for study because it produced the
highest quantities of taxol/l and because there is a large body of literature dealing
with the genus PenicilHum, including examples of transforming this fungus. For
these reasons the work described in chapters 3 and 4 was performed.
The preliminary work that led to chapter 2 also began as an investigation of
the enzymatic step involving phenylalanine-2, 3-aminomutase in the biosynthesis
of taxol. There was no phenylalanine-2, 3-aminomutase activity detected, but
aromatic L-amino acid decarboxylase (AADC) activity was detected. AADC
activity is associated with the production of biologically active compounds in
other organisms, as described in chapter 1. An AADC had not been described
from a fungal source, even though fungi produce compounds that are most likely
135
derived from AADC activity (see figure 12). The work presented in this thesis
demonstrated that a fungus did produce an AADC, although at this time the
enzyme cannot be directly linked to the production of any of the compounds
isolated from it (see figure 1). The biological function of AADC in P. raistrickii
isolate H10BA2 is one aspect that future research can address. One experiment
would be the isolation of the gene coding for this enzyme. There are several
methods that can be used to acquire the gene, including utilization of polymerase
chain reaction primers described in the literature which have been used to isolate
these genes out of cDNA libraries from Papaver somniferum (36). Another
method would involve determining the amino acid sequence of the protein or a
portion of the protein and then developing degenerate DNA probes for the
screening of DNA libraries of the fungus. Once the gene has been isolated and
cloned, it can be used to disrupt the AADC gene within P. raistrickii via
homologous recombination, as described in chapter 1. Homologous
recombination has been described in P. chrysogenum (49). An isolate of P.
raistrickii that did not have AADC activity could then be used to determine the
biological function of AADC within the fungus. If an AADC lacking mutant of P.
raistrickii was unable to produce certain secondary metabolites produced by the
wild type P. raistrickii, then the enzyme is involved in the production of those
secondary metabolites. Homologous recombination would allow for the
development of an AADC lacking mutant, which would be better than an AADC
lacking mutant derived from random mutagenesis techniques, such as use of UV
136
irradiation or chemical mutagens. Mutants that would be derived from this
technique could have mutations at a variety of locations within their genome.
This would make correlation of loss of secondary metabolite production to lack of
AADC activity less direct.
Experiments to detect the presence of AADC activity in another isolate of P.
raistrickii were performed. No detectable AADC activity was present in P.
raistrickii ATCC # 48678 when grown and processed as P. raistrickii isolate
H10BA2 was. This does not mean that the fungus does not contain an AADC.
P. raistrickii ATCC # 48678 may contain such an enzyme, but it could be
produced under different conditions. P. raistrickii AJCC # 48678 may be
considered an AADC minus isolate of P raistrickii when grown under specific
conditions. P. raistrickii ATCC # 48678 does not produce detectable quantities of
taxol (85) and its ability to produce other compounds has not been investigated.
P. raistrickii isolate H10BA2 does produce taxol and AADC. A direct correlation
between taxol production and AADC Activity should not be made given this
information. The true genetic relatedness between P raistrickii ATCC # 48678
and isolate H10BA2 is not known. These two fungi were considered to belong to
the species P. raistrickii because they fit into that category according to
morphological characteristics, not by direct genetic analysis. General
comparisons of these two isolates can be made, but direct comparison of gene
function requires more information on the genetic relatedness of these two fungi.
It can be stated that AADC activity is not ubiquitous in the species P. raistrickii
137
when two isolates are grown under identical conditions, and that AADC activity
may be correlated to secondary metabolite production in specific strains.
Some of the possible functions of AADC in P. raistrickii are the production
of biologically active secondary metabolites, or the production melanin. Though
no reports of fungal melanin originating from the amines of aromatic amino acids
has been described (39) this pathway does exist in insects (38) and may possibly
occur in fungi.
Another possibility would be to determine if AADC activity could be detected
in those fungi that produce alkaloids derived from aromatic amino acids. These
fungi include Psilocybe spp., Claviceps purpurea, Amanita spp., Boletus
erythropus, Sarcodon imbricatum, Coprinus spp., Panaeolus spp., Conocybe
spp., and Stropharia spp. (23). The AADCs from these fungi could then be
subjected to the same experiments described above for the AADC from P. .
raistrickii isolate H1QBA2.
A potential application of a fungal AADC includes using the gene encoding
for this activity as a selectable marker in fungal transformation experiments.
The
Catharanthus roseus AADC sequence has been used as a selectable marker in
plant transformation experiments (66). Another application of a fungal AADC
would be in the production of the anti-cancer compounds vincristine and.
vinblastine, which are produced in Catharanfhus roseus from tryptamine.
One
scenario would be the introduction of other key plant genes for the biosynthesis
of these compounds into a fungus, which already produces the necessary
138
precursors, to make vincristine or vinblastine. Large fermentations of such a
fungus could then be used to produce these compounds.
Transformation experiments with P. raistrickii isolate H10BA2 were
performed to develop the techniques necessary for manipulating genes involved
in taxol biosynthesis to increase taxol production. Several species within the
genus Penicillium have been transformed with plasmid DNA, including P. urticae,
P. chrysogenum, P. roqueforti, P. citrinum, P. paxilli, P. canescens, and P. freii
(47, 49, 52, 53, 54, 57, 55). Preliminary experiments in the transformation of P.
raistrickii isolate H10BA2, with the plasmid pAN71, demonstrated that
hygromycin B resistant colonies could be isolated. Some of these colonies were
placed on non-selective media, and the conidia that arose under non-selective
conditions gave rise to colonies when transferred to selective media. This
demonstrated that P. raistrickii has the potential to be transformed. The definitive
experiment of performing southern blot analysis of putative transformants has yet
to be done.
Transformation of P. raistrickii isolate H10BA2 would allow a variety of
experiments to be performed. One experiment that I proposed to do would be
the introduction of human MDR 1 into the fungus. Human MDR1 codes for a PgIycoprotein that is responsible for the multidrug resistance phenotype present in
some cancer cell lines (93). P-glycoprotein acts as a pump to keep potentially
toxic compounds out of a cell (94). P-glycoprotein is able to pump out a variety
of compounds from cells that are selected for resistance to a single compound,
139
thereby resulting in a multidrug resistance phenotype (94). Introduction of Pglycoprotein into a taxol producing microorganism could result in the transfer of
taxol or its precursors out of the cell and into the medium. This would allow
easier downstream processing of microbially produced taxol since the medium,
not the biomass, would be processed for taxol isolation and purification. The
removal of intracellular taxol could also increase the production of taxol if the
biosynthetic pathway is controlled by feed back inhibition.
The investigation of taxa-4(5), 11(12)-diene synthase from the fungus P.
raistrickii isolate H10BA2 was an attempt to isolate the first and potentially most
critical enzyme in the biosynthetic pathway to taxol. As described in chapter 4,
taxadiene synthase activity was not identified when the techniques used to
identify the enzyme from the yew tree were applied to the fungus. If future
experiments to find taxadiene synthase within this fungus are considered then I
would recommend not trying to identify the enzyme as I have described. One
thing to do would be to avoid exposure of protein solutions to glass. It was found
that exposure of protein solutions with AADC activity to glass resulted in a loss of
enzyme activity. This is difficult to do because the enzyme assays for taxadiene
activity must be performed in glass since the substrate binds to plastic. There
may be some plastics to which the substrate does not bind and these would be
necessary for such a study. The first goal should be to separate taxadiene
synthase activity away from phosphatase activity. This may involve using other
chromatography techniques such as cation exchange or hydrophobic interaction.
140
It is interesting to note that optimal enzyme activity for AADC and phosphatase
(see figures 28 and 44, respectively) occurs at pH 5.5. This may indicate that the
intracellular pH within P. raistrickii isolate H10BA2 was around pH 5.5.
Experiments should involve assaying for enzyme activity from pH 5.5 to 8.5.
A different approach to the identification of a taxadiene synthase would be
to use DNA based technology to identify the gene for the enzyme. A cDNA clone
for taxadiene synthase from the yew tree has been reported (95). The
techniques used to identify the yew gene may be used to identify the fungal
gene.
The basic assumption made at the beginning of the work on fungal
taxadiene synthase was that the fungal enzyme would be very similar to the yew
enzyme. The results of chapter 4 indicate that the fungal enzyme may not be
similar to the yew enzyme since the techniques used to identify the yew enzyme
did not work when applied to the fungal system. One of the reasons the
assumption was made was based on the hypothesis that horizontal gene transfer
could have occurred between the fungus and the tree, resulting in a transfer of
the genes needed to synthesize taxol. Horizontal gene transfer is the transfer of
DNA between species or kingdoms. Horizontal gene transfer occurs within the
prokaryotic kingdom between species and results in the transfer of plasmids
coding for drug-resistance. One example of horizontal gene transfer between
kingdoms is crown gall of grapes caused by Agrobacterium tumefaciens (96). In
this scenario the bacterium, Agrobacterium tumefaciens, transfers a Ti plasmid
141
into grape cells. The Ti plasmid integrates into the cell’s genome and expression
of genes on that plasmid results in the formation of auxins and opines. The
auxins cause the plant cells to divide and form a gall that provides an optimal
environment for bacterial growth. Opines are utilized by the bacteria as a carbon
and nitrogen source. There is one report of horizontal gene transfer between a
mite and a fruit fly (97). Isolation and characterization of a fungal taxadiene
synthase would have allowed comparison to the yew taxadiene synthase. This
comparison could have demonstrated if a horizontal gene transfer event did take
place between the two organisms allowing for transfer of taxol biosynthetic
pathway genes.
142
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