JOWAL
OF BIOLOGICAL
CHEMISTRY
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
%E
Vol. 269,No. 48,Issue of December 2, pp. 3072630733, 1994
Printed in U.S.A.
Triple Hydroxylationof TetracenomycinA2 to TetracenomycinC in
Streptomyces glaucescens
OVEREXPRESSION OF THE tcmG GENE IN STREPTOMYCES LIVZDANS AND CHARACTERIZATION OF THE
TETRACENOMYCIN A2 OXYGENASE*
(Received forpublication, March 28, 1994, and in revised form, July 29, 1994)
Ben She& and C. Richard Hutchinson-SPn
From the $School of Pharmacy and the SDepartment of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706
Nucleotide sequence analysis of the tcmG gene has have elucidated all of its biosynthetic intermediates (Fig. 1 B )
suggested that the TcmG protein is responsible for the (9-11). We also characterized severalkey enzymes of the pathtriple-hydroxylation of tetracenomycin
(Tcrn)A2 to Tcm way, including the Tcm F2 polyketide synthase (12-141, the
C in Streptomyces glaucescens(Decker,H., Motamedi,H., Tcm F2 cyclase (151, and the Tcm F1 monooxygenase (16) and
andHutchinson, C. R. (1993) J. Bacterial. 175, 3876- cloned (17) and analyzed (18-23) the nucleotide sequences of
3886). The heterologous expressionof the tcmG gene in the complete gene cluster for the biosynthesis of 1 (Fig. 1B).
Streptomyces lividans and the purification and charac- These studies have
provided detailed insightsinto the biochemterization of TcmG protein, which we have named
Tcm istry and genetics of the biosynthesis of 1 in S. glaucescens,
A2 oxygenase, are described here. NH,-terminal amino which canserve as a model for the formation of aromatic
acid analysis of the purified enzyme ledto the revision polyketides in general. During this work, we have suggested
of the translational startsite of tcmG to a TTG, 33 base that the three cis-hydroxy groups at the 4, 4a, and 12a posipairs downstream ofthe GTG site assigned initially on tions of 1 are introduced by hydroxylation of Tcrn A 2 , 6,an
the basis of nucleotide sequence analysis. Tcm A2 oxy- unprecedented process catalyzedby the TcmG protein (20). The
genase is a monomeric proteinin solution and contains latter idea was further supportedby complementation experi1 mol of non-covalently boundFAD; the apoenzyme can ments with the Tcm C non-producing mutant S. glaucescens
be partially reconstituted in vitro by addition of FAD. WMH1089 that containsa 180 base pairdeletion mutation that
Tcm A2 oxygenase exhibits an optimal pH of 9.0-9.5 and has been mapped to the
tcmG gene (20); the production of 1 was
prefers NADPH over NADH as an electron donor. The restored upon transformation of the WMH1089 strain with
apparent K‘, of the enzyme for Tcm A 2 , NADH, and
pWHM126 that carries most of tcmG (20). Moreover, the results
NADPH are 1.81 f 0.38,260 f: 19, and 82.1 * 17 m,respeconly the
tively, andthe apparent V’=-for the reaction is 14.7 * 1.1 of in vivo “0,feeding experiments have indicated that
nmol Tcm C/min-mg. Purification and characterization 4- and 12a-OH groups of 1 are derived from molecular oxygen,
of Tcm A2 oxygenase provide direct
evidence to support leaving the 4a-OH to arise presumably from water (6).
the notion thatthe angular hydroxy groups of naphtha- To extend our investigation of the biosynthesis of 1, in parthe underlyingenzymatic
reaction
cenequinones likeTcm C are introducedfrom “O, via a ticulartounderstand
mechanisms of the pathway, we studied thehydroxylation of 6
mono- or dioxygenase process.
t o 1 in vitro and report here the overexpression of tcmG in
Streptomyces lividans and the purification and characterization of the Tcm A2 oxygenase. NH,-terminal amino acid analTcm’ C, 1, a polyketide antitumor antibiotic, wasfirst
ysis of the purified enzyme has led to a revision of the tcmG
isolated from Streptomyces glaucescens in 1979 (1) and then translational start site to a TTG codon, 33 base pairs downreisolated from Streptomyces H-881 in 1984 (2). Its absolute stream of the GTG codon assigned initially on the basis of
stereochemistry was established in 1992 (3) as the 4-R, 4a-R, nucleotide sequence analysis (20). Our results establish the
12a-R configuration (Fig. lA). Together with elloramycin (4,5), stoichiometry of the conversion of 6 to 1 and prove that this
2, tetracenomycin X (61, 3,dutomycin (71, 4, and viridicatum- reaction is catalyzed by the enzymeencoded by tcmG. Tcm A2
toxin (8),5 , l forms a small groupof naphthacenequinones with oxygenase was found to be a monomeric flavoprotein containunique structural featureof the highly hydroxylated semiqui- ing 1mol of non-covalently bound FAD and to requiremolecunone moiety (boxed in Fig. lA).
lar oxygen and reduced nicotinamide cofactors.
We have been studying the biosynthesisof 1 as a model for
EXPERIMENTAL PROCEDURES
the family of polyketides with fused aromatic rings. Previously,
we established that 1 is formed from acetate and malonate and General-UV-VIS spectra were recorded on a Hitachi U-3000 spectrophotometer (SanJose, CA). Refrigerated centrifugationwas done in
a Sorvall RC-5B superspeed centrifuge (Newtown, CT). A Pharmacia
* This work was supported by National Institutes of Health Grant FPLC system was used for enzyme purification and all FPLC columns
CA35381. The costs of publication of this article were defrayed in part were purchased from Pharmacia Biotech Inc. HPLC was done with a
by the payment of page charges. This article must therefore
be hereby Waters model 501 pump system (Marlborough,MA) and a Waters 484
marked“advertisement”inaccordancewith
18 U.S.C.Section 1734 variablewavelengthabsorbancedetector.Enzymeincubations
were
solely to indicate this fact.
ll To whom correspondence should be addressed: School of Pharmacy, performed in a GCA Precision shaking waterbath (+ 0.1 “C) (Precision
in a rotary
425 N. Charter St., Madison WI 53706. Tel.: 608-262-7582; Fax: 608- Scientific Inc.,Chicago, IL). Fermentations were carried out
shaker-incubator (Series
25, New Brunswick ScientificCO.Inc., Edison,
262-3134; E-mail: CRHUTCHI@FACSTAFF.WISC.EDU.
The abbreviations used are: Tcm, tetracenomycin; BSA, bovine se- NJ). Analytical TLC was done on precoated Keiselgel60 SiO, FZs4glass
rum albumin;DTT, dithiothreitol;FPLC, fast protein liquid chromatog- plates (0.25 mm) and was visualized by long-and/orshort-wave
raphy; HPLC, high performance liquid chromatography;RBS, ribo- UV light.
S. glauBacterial Strains, Plasmids, and OtherMaterials-The
some-bindingsite; PAGE, polyacrylamide gel electrophoresis; TLC,thin
layer chromatography;kb, kilobase(s);PCR, polymerasechain reaction. ceseens type strains (91,S. liuidans 1326 (241, and plasmid pWHM3 (25)
30726
Tetracenomycin A2 oxygenase from S . glaucescens
4 R = sugars
B
acetyl CoA
TcmJKLMN
+
30727
5
TcmI
t
9 malonyl CoA
/
CO2H
OH
OH
OHOH
CH3
CH3 0
/
OH
0
-
HO
-
TcmH
TcmNOP
OH
OH
CH3 0
OH
TcmG
1
CH, 0
OH
6
FIG.1. Tcm C and related naphthacenequinones (A) and biosynthetic pathway of Tcm C in S. glaucescens ( E ) .
are described elsewhere; pSP72 andpGem7zf were obtained from Pro- CCGCCAGCGGGCTCCCTC-3', 1.0 ng), 5 pl of formamide, 1p1 of BSA
mega Corporation (Madison, WI). The ermE+ promoter containing plas(1mg/ml), 36 p1 of H,O. The reaction mixture was covered with three
mids pWHM63, pWHM64, and pWHM65 were gifts from G. Meurer.'
drops of mineral oil, boiled for 5 min, and placed at 70 "C. To this
Thiostrepton was obtained from Sal Lucania
at the Squibb Institute
for
mixture was added 4 pl of the dNTP mixture (final concentrationsfor
Medical Research (Princeton, NJ). Unless
specified, common chemicals, dCTP and dGTP were 60 p and for dATP and dTTP, 40 p ~ respec,
restriction enzymes, DNA ligase, and other materials for recombinant tively) and 1 pl of Taq polymerase (4.5 units). The PCR temperature
DNA procedures were purchased from
standard commercial sources
program was as follows: 24 cycles of 40 s at 96.5 "C and then2.5 min at
and used as provided.
71 "C; after thelast amplification cycle, 40 s at 97 "C and then7 minat
DNA Isolation a n d Manipulation-Plasmid DNA from Escherichia 71 "C. After cycle 12, a n additional 0.5 pl of Taq polymerase (2.25 units)
coli was prepared according to
Lee and Rasheed (26). For plasmid
DNA was added. The final amplified 306-base pair fragment was purified
by
isolations from Streptomyces spp.,
the cells were lysed according to
agarose gel electrophoresis, digested with HindIII and SacI,
and ligated
Hopwood et al. (271, then treatedas described by Lee and Rasheed (26). with the4.2-kb HindIII-SstI fragment obtained
from partial digestionof
Agarose gel electrophoresis, restriction enzyme digestion, DNAligation,pWHM1018 to yield pWHM69. The tcmG gene was transferred from
and preparationof competent E.coli DH5a cells and their transforma- pWHM69 as a SphI-XbaI fragment into similar sitesof pWHM64 and
tion were performed by established methods (28). DNA was purified
pWHM65 to give pWHM70 and pWHM71, from which the 2.1-kb EcoRI
from agarose gels with theQIAEX kit asdirected by the manufacturer fragments were cloned into the same siteof pWHM3 to yield pWHM72
(QIAGEN Inc., Chatsworth, CAI. Protoplasts of Streptomyces spp. were and pWHM73, respectively (Table I).
prepared and transformedby the methods of Hopwood et al. (27).
Protein Analysis-Protein concentrations were determined by the
Construction of pWHM68, pWHM72, and pWHM73-To prepare Bradford (29) method withBSA as the calibration standard. Pure
Tcm
pWHM68, a 2.1-kb EcoRI-Hind111fragment frompWHM1018 (20) that A2 oxygenase also was quantifiedby UV absorption a t 280 nm where
contains tcmG was cloned intothesamesites
of pSP72 t o give
the molar absorbance index (ez8,, ",,I is 75.7 mM-l.cm-l (this value was
pWHM66. The later was digested with HindIII and
EcoRV and the
calculated from the amino acid sequence deduced for the apoenzyme
resulting 2.1-kb fragment was cloned into the HindIII-SmaI sites of from tcmG). The molecular weight of the enzyme subunit was deterpWHM63 to give pWHM67, from whichaNsiI-XbaIfragmentwas
mined by SDS-PAGE using theLife Technologies, Inc. protein molecular
moved into the XbaI-PstI sitesof pWHM3 to yield pWHM68 (Table I). weight standards of myosin H-chain 200,000, phosphorylase b 97,400,
To construct pWHM72 and pWHM73, polymerase chain reactions
BSA 68,000, ovalbumin 43,000, carbonic anhydrase 29,000, p-lacto(PCR) were used with site-specifically
modified oligodeoxynucleotides to globulin 18,400, and lysozyme 14,300. SDS-PAGE was performed acgenerate a fragment of the NH, terminus of tcmG with a unique SphI cording to the method of Laemmli (30) or on the PhastSystem (Pharsite at an ATG translational start site. PCR was carried out with a macia) as describedby the manufacturer and thegels were Coomassie
Perkin Elmermodel 480 DNA thermal cycler (Oak Brook, IL) withTaq Blue-stained (31). The abundance of each band was then quantified
polymerase and buffersupplied by PromegaCorporation. Oligode- on a MolecularDynamics
model 300A ComputingDensitometer
oxynucleotide primers were synthesized on a model 391 DNA synthe- (Sunnyvale, CA). The molecular weightof the native TcmG was detersizer (Applied Biosystems, Foster City, CA), purified on 8 M urea, 16% mined by gel filtration chromatography on a Superose 6 HR 10/30
polyacrylamide gels, and electroeluted from the gel slices. The PCR
column in 20 m~ sodium phosphate, pH 7.2, 1 mM DTT, 150 mM NaCl
mixture consistedof 50 p1 of the 2 x buffer (40mM Tris-HC1, pH 8.3,2.4
with a flow rate of 0.4 ml/min and the column was calibrated with blue
mM MgCl,, 40 mM KCl, 0.2% Triton X-100),2 plof DNA (20 ng), 1 pl of
dextrin 2 x lo6, alcohol dehydrogenase, 150,000, BSA 66,000, carbonic
3'
primer
(5'-ACACCAAGCTTCTAGAGCATGCCCGTTTCCGACCG-anhydrase 29,000, and cytochrome c 12,400 purchased from Sigma.
ACCGAAA-3', 1.0 ng),1pl of 5' primer (5'-AATTGGGAA?TCGAGCTCEnzyme Assays-The substrate 6 and authentic product 1 were iso-
' G. Meurer, and C. R. Hutchinson, unpublished data.
lated from S . glaucescens WHM1089 and S. glaucescens GLA.0, respectively, and characterized as described elsewhere (1,9, 10).
Tetracenomycin
30728
A2 oxygenase from S. glaucescens
TABLEI
Plasmids used in this study
Plasmid
Description
Refs
___
pWHM1018
pWHMlO19
pWHM63
pWHM64
pWHM65
pWHM66
pWHM67
pWHM68
pWHM69
pWHM70
pWHM71
pWHM72
pWHM73
tcmG gene behind the tcmG promoter in pUC19
tcmG gene behind thetcmG promoter in pWHM3
ermE* promoter in pGem7zf
ermE* promoter engineered with theRBS of CCCAGGAGGT
complementary to the 3'-endof 16 S rRNA of S. liuidans in pGem7zf
ermE* promoter engineered with theRBS of GAAAGGAGGT
found in the melCl gene from S. antibioticus in pGem7zf
tcmG gene behind the tcmG promoter in pSP72
tcmG gene behind the tandem ermE* and tcmG promoters in pGem7zf
tcmG gene behind the tandemermE* and tcmG promoters in pWHM3
tcmG gene in pUC19
tcmG gene behind theermE* promoter with theRBS of
CCCAGGAGGT in pUC19
tcmG gene behind the ermE* promoter with the RBS of
GAAAGGAGGT in pUCl9
tcmG gene behind theermE* promoter with the RBS of
CCCAGGAGGT in pWHM3
tcmG gene behind theermE* promoter with theRBS of
GAAAGGAGGT in pWHM3
20
20
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
Typically, 250 pl of assay solution with10% (v/v) dimethyl sulfoxide,
1 h and centrifugedas above to
sulfate. The suspension was stirred for
consisting of 100 p~ 6, 250 p~ NADPH, and 1 mM DTT in 50 mM remove the precipitate. The resulting supernatant was brought
to 62%
ethanolamine-HC1 buffer, pH 9.5, in thepresence of enzyme (10-50 pl), saturation (375 ghiter) with
solid ammonium sulfate and was stirred
for
was incubated at 25 "C. The assay was initiated by addition of 6 and
1 additional h. Centrifugationas above afforded a pellet that had the
terminated by addition of solid NaH,PO, to saturation and extraction enzyme activity.
with EtOAc (2 x 400 pl). The EtOAc extracts were collected and conStep 3. Sephacryl S-200 HR column: the ammonium sulfate pellet
dissolved in 50-120
was dissolved in a minimumvolume of 20 mM sodium phosphatebuffer,
centrated in oacuo to dryness, then the residue was
p1 of methanol and analyzed by TLC or HPLC. SiO, plates were used pH 7.2, 1 mM DTT, 150 mM NaCl and applied to a Sephacryl S-200 HR
and developed in CHClfleOH (955, v/v); under these conditions6 and
column (2.6x 60 cm). The column was eluted
at the flow rate of 2 mumin
1 have a n Rfof 0.77 and 0.28 and, underUV light, display a character- with the samebuffer, and 5-ml fractions were collected.
istic yellow and blue fluorescence, respectively. This TLC method was
Step 4. Mono Q HR 10/10 column: fractions containing the enzyme
used throughout the purification to monitor the enzyme activity quali-activity after gel filtration chromatography were dialyzed against 25
tatively. Alternatively, a HPLC method was developed that provided a m~ Tris-HC1, pH 8.0, 1 m~ DTT and applied to a Mono Q HR 10/10
mM Tris-HC1 buffer, pH 8.0, 1 mM D m ,
quantitative analysis of the enzymatic synthesis of 1 from 6. Assay column. After washing with 25
samples were analyzed by HPLC on a Nova-Pak C,, column (Waters) the column wasdeveloped a t a flow rate of 2 mVmin with a linear 60-ml
developed with a linear gradient fromCH,CN/H,O/AcOH (20:80:0.1%, gradient from 0 t o 0.6 M NaCl in the same buffer, and 2-ml fractions
v/v) t o CH,CN in 10 minfollowed by additional 5 min at 100% CH,CN
were collected.
at a flow rate of 2 mumin with
UV detection at 280 nm. Thecolumn was
Step 5. Alkyl Superose HR 5/5 column: the active fractions after
calibrated with authentic 1 and 6 that, under these conditions, have
anion exchange chromatography were brought to1.2 M ammonium sulfate by addition of solid ammonium sulfate and were applied
to a n Alkyl
retention times of 6.5 and 12.0 min,respectively.
The HPLC assay method was used in all studies with the
following Superose HR 5/53 column. Thecolumn was washed with50 m~ sodium
phosphate, pH7.2,l mM DTT, 1.2 M (NH,),SO,, then developed at a flow
modifications. For the pH dependence study, the assays were performed
from 1.2 to0 M (NH,),SO,
in 50 mM Tris-HC1 buffer, pH 6.5-9.0, and 50 mM ethanolamine-HCl rate of 0.5 mumin with a linear 15-ml gradient
buffer, pH 8.0-10.5, respectively, in thepresence of 11.2 pg of TcmG.For in the samebuffer, and 0.5-ml fractions were collected. The final prepof active enzyme was stored
at -20 "C, and no significant loss of
determination of the kinetic parameters, the assays were done with aration
the
concentration of 6 varied from 0.5 t o 30 PM, 1.0 mM NADPH, and 2.93 pg enzyme activity wasobserved over a 4-week period.
NH,-terminal Sequence Determination-A portion of the purified
of TcmG, or with concentrationsof NADH or NADPH varied from 50 to
1.5 mM, 100 p~ 6, and 5.85 pg of TcmG, respectively, in 50 m~ ethanol- TcmG protein (16 pl, 20 pg) was loaded onto a ProSpinm sample prepamine-HC1 buffer, pH 9.0, for durations that yielded a linear relation- aration cartridge (Applied Biosystems) and washed according to the
ship between product formation and time. The apparent kinetic con- instructions provided by the manufacturer. A fraction of the protein
bound-polyvinylidene difluoride membrane
of the ProSpinTMdevice was
stants of K
'
,and V m U were determined by a nonlinear regression
used directly for the NH,-terminal amino acid sequence determination
analyses (32) basedon the Marquardt-Levenberg algorithms.
at theUniversity of
Enzyme Purification-All steps were carried out a t 4 "C except for by automatedEdmandegradationchemistry
Wisconsin
Biotechnology Center (Madison, WI).
the brief time when the enzyme was on the FPLC columns that were
at
Prosthetic Group Determination-Pure TcmG protein (200 pl,
224 pg)
room temperature.
in 25 mM Tris-HC1, pH 8.0, was boiled for 5 min, cooled on ice immediStep 1. Preparation
of cell-free extract: culturesof S. liuidans transformed with the tcmG expression plasmids were grown in R2YENG ately, and centrifuged i n a nEppendorff centrifuge for 20 min to pellet
the denatured protein. The supernatant was subjected to HPLC analmedia(9,17)withthiostrepton
(10 pg/ml) in 2-liter
a
baffled
by
Erlenmeyer flask. After incubation
at 30 "C and 300 revolutions/minfor ysis on aC,, column to quantify any flavin prosthetic group released
3 days,cells were harvestedby centrifugation (13,600x g , 20 min, 4"C) this process (33). The column was calibrated with FAD and FMN, reand washed sequentially with0.5 M NaCl and 0.1M sodium phosphate spectively, and was developed with the following program: flow rate of
buffer, pH 7.2, with centrifugation as necessary to yield approximately 2 mumin withUV-VIS detection at 450 nm in5 mM NH,OAc buffer, pH
15 g cellsfliter (wet weight). The washed cells were suspended min
M 1006.5, 10% MeOH (v/v) in the first 5 min followed by a 20-min linear
sodium phosphate buffer, pH 7.2, 2 mM DTT, 0.1 mM phenylmethylsul- gradient from 10 to 70% MeOH in the samebuffer. Under these condiof 10.8 and 11.5 min, respecfonyl fluoride, 1 mM EDTA, 10% glycerol (10 mug cells). Lysozyme (1 tions, FAD and FMN have retention times
mg/ml) was added, and the mixture was left to incubate atroom tem- tively (34). The concentrationsof FAD and FMN were determined acof,"%:E
= 11.3 mM-'.cm" and
perature for 2 h.To this viscous slurry, solid MgCl, (5 mg/ml) and DNase cording t o their molar absorbance indexes
= 12.2 m-l.cm-l, respectively.
(1pg/ml) were added. The resulting slurry was incubated
on ice for 1h, ,~?:E
Preparation of Apo-Tcm A2 Oxygenase andItsinVitro
Reconand cell debris were removed by centrifugation (27,500 x g , 20 min,
stitution-To prepare apo-TcmG, 1 ml of pure TcmG protein (1.46 mg)
4 "C) to yield a cell-free extract. The enzyme activity in preparations
from this and succeeding purification steps was followed by the TLC was dialyzed against200 ml of 100 mM potassium phosphate buffer, pH
4.0, 2.0 M KBr for 2 days with four buffer changes (35, 36). This sample
assay method.
was thendialyzed against200 ml of 25 m~ Tris-HC1, pH 8.0, 1mM DTT
Step 2. Ammonium sulfate fractionation: the
cell-free extract was
brought t o 41% saturation (234 gfliter)by addition of solid ammonium for 2 days with five buffer changes to yield the apoenzyme that was
Tetracenomycin A2 oxygenase from S. glnucescms
A
1
KDa
- 200
KDa
2 0 ,
97.4.
68
-
- 97.4
TcmG
-
T.w.t: 11
B
1 2 3 4 5 6
- 68
29 -
30729
Purifirntion of tlw 7i.m A2 o.r.vpnr1.w fmm S . I1r~rtlr1n.s/p\\'\f.\l681
Data were ohtained from 13 g r w c s t weight I c r l l s . Thc complete a s w y
solution of 250 pI with 10'; I V / V Idimrthyl s u l f o x i d r ~ronsistrd nf lOcl
6, 250 p51 NADPH. and 1 m v I)TT In 50 m u ~ ~ t h : ~ n r ~ l : ~ m i n c ~t u-llflf(w' l,
pH 9.0, in t h r prrsence of 5.85 )IC of Tcm(;. a n d \ v a s ~nc~rh:~tc.d
;It 25 ('
for 10 min thenanalyzrd
hy t h r llI'l,(' mr~tt111dt l r w m t w d u n d w
2
.TcmC
"Experimental Procedures."
-
~
43
18.4.
14.3.
- 43
~
I+ot(*in / w f l v l t v
,,,p
,l"l#,l
rnfn
86.5
Cell-free rxtract
(NH,l),SO,pellets
Sephacryl 5-200
570
312
Mono Q H R 10/10
Phrnyl Superow HR 515
"~
FIG.2. Ovcw-xprrssion of the tcmG gene in S. lividann. A, SDSPAGE (7.5'; I :111;11ysisof 500 pg of cell-free extracts from S. Iiuidnns
containing pWll.llW Ilnnr I ), pWHM73 tlnnr 2 ) . pWJJM72 (Innr 3 1 ,
pWHM 10 19 (Innr 4 I, and pWHM3 (Innr 5 1, respectivrly; lune 6 contains
molecular weight standards ( s w "Experimental Procedures"). R , SDSPAGE (12.W J'hast gel) analysis of the purified Tcm A2 oxygenase (lnnr
2 ) : Innr I contains molecular weight standards.
-
"
"
S1rp
~~~
~
81.9
32.2
I8H
162
166
1.10
I:iH
~
~Y ~ ~ , I ,~
I
rrn,r,l
;
';
r/r,l,l
nt&*'mr~t
0.217
l1.2H.4
0.532
1.71
4.29
;
~~t~r~~r:~,~,trl
100
Hfi.2
H H 3
7.1.5
7:!,4
1.oo
1.31
2:15
H.1G
1:I.H
~
approximatelv 5-fold higher lrvrl of TcmG than any singlrpromoter counterpart (Fig.2A, ahundancr in lanrs 1-4 = 4.8. 1.0.
0.89, 0.89).
Purifirntion of thr
TrmC
Prntcin from S . 1ir.irIlnns
IpWHM6iR)"Since S . lirlidans rpWHM681 rxprrssrdtrmG most
effectively among the constructs trsted,a crll-frrr extract \vas
assayed to determine the residual enzyme activity. For in oifro reconstitution ofthe apoenzyme. a 500-pl solution containing 1.65 nmol ofthe prepared from this recombinant strain for the purificntitrn of
was fractionatrd hy addition of
apoenzyme, 16.5 nmol of either FAD or FMN, respectively, in 25 mM TcmG. Theenzymeactivity
a t 41-62'; sat.urntion. and
Tris-HCI, pH 8.0. 1 m y DTT was incuhated on ice for 1.5 h (35,361;1.65 (NH,,),SO., to the cell-frre extract
nmol of the holoenzyme w a s similarly treated with FAD or FMN a s
more than RFir> of the enz.yme activitv was recovrrrtl in t h r
controls. BothFAD and FMN were purified by HPLC on C,, column (NH,,),SO., precipitatedpellet.Furthrrpurificationwasprrunder the conditions described above to ensure no contamination of formed as descrihed undrr "Exprrimental Procrdurrs" hv sizr
other flavin derivatives in the commercial materials (34). The resulting
reconstituted enzyme was assayed directly hy the HPLC method with- exclusion tSephacryl S-200 HRI. anion-exchangr (Mono Q H R
10/10), and hydrophohic interaction (Alkyl Suprrosr
HR 5 / 3 1
nut attempting to remove the excess FAD or FMN.
chromatography. Thew procrdurrs gave purr TcmG\vith an
RESULTS
overall 20-fold purification in more than 70r; yirld fTahlr 111
(approximately 40 mgllitrrof isolated yirld,. Thr purifird proHeterologous Exprrssion of thr tcmG Gene in S. lividanstein was homogeneous when examined hy SDS-PAGE wherr it
Since Tcm A2 oxygenase activity was not detected in cellular
extracts from either theS. glaucmcrns GLA.0 wild-type strain migrated as n single hand of 60.000 Da I Fig. 213 I.
NH,-trrminnl Srqurncr Drtrrminntion-To confirm that thr
or the WMH1094 (20)Tcm C non-producing mutant that hiotransformed 6 to 1 effectively in vivo, we expressed the tcmG isolated protein whose purification was guitlrd hy Tcm A 2 oxygene in S. 1ividan.s to facilitate the isolation and characteriza- genase activity was indeed the trmG product. a portion of t h r
purified protein was suhjected to amino-trrminal srqurncing
tion of Tcm A2 oxygenase.
pWHM1019,
pWHM72,
and
w a s foundto
hc,
pWHM73 were made in the highcopy number vector pWHM.7 and,tooursurprise,theNH,terminus
(25) so as to place the expression of tcmG under the control of STEEVPVLIV-, which is 12 amino acids shortrr than that prrtcmG RBS fromS. glaucescens dicted from the tcmC sequence (Fig. 3 A ) (201.Although itis
t h e tcmG promoter (21) with the
(20) or of t h e ermE* promoter (12) in combination with a RRS known that post-translational processingof thv nnscrnt protrin
from either Strrptom.yces antihioticus (37) or S. lioidans (.78), occasionally can result in loss of a short prptidr frapnrnt 1.10.
since it is known that theermE" promoter displays the strong- 41), re-examination of the tcmC sequencr rrvralrd a possihlr
7 ° K ; that is 88 hasr pairs
est activityamongseveralcommon
Streptomyces promoters new translational start site, thr
studied (39) and that the sequenceof the RBS can also have a downstream of the original GTG translational start sitr (Fig.
3 A ) (20). This'ITG is preceded by a putativr RRS of GAAA(;distinct effect on t h e level of gene expression." pWHM68 was
constructed to examine the effect
of placing the rrmE'" and GCTGC, which compares flavorahly with GAAA(;GA(XT that
of 16 S rRNA of S . 1it.idnns ( 3 8 I
tcmG promoters in tandem on the levelof expression of tcmG. is complementary to the 3'-end
All four plasmids were introduced by transformation into S. and predicts an NH, terminus idrntical to thr onr tlrtrrminrd
from the purified TcmG protein (Fig. 3A I rxcluding thr nlrt
Iioidans 1326 (24), and the levels
of tcmG expressionwere
residue. Therefore, we revised the translational start sitr
of
assayed by SDS-PAGE of cell-free extracts prepared and analyzed as described under "Experimental Procedures." A distinc-tcmC to this I T G codon, and Fig. 8B shows thrdrducerl amino
NH, terminus ofTcmG.in fact.
a size of 60,000 Da was observed in all acid sequence ofTcmG. The new
tive hand migrating with
2 A , lanes 1-4); this band was absent in
a aligns well with several othrr hactrrial oxygrnasrs 1201.Sincr
fourcases(Fig.
sample from the control culture of S. lividans (pWHM3) (Fig. the amino terminusof the isolated protrin was found to hr Srr.
2A, lane 5 ) . The apparent size of the expressed protein is con- it can he calculated from the rrvisrd nucleotidr srqurncr that
of 60,429 D:I with
sistent with the size
of 61,694 Da predicted from the nucleotide the Tcm A2 oxygenasr has a molrcular mnss
sequence of tcmG (20). Whereas the abundance of TcmG was a n PI of 5.57.
Molecular Wpight Drtrrminathn-Thr nativr form ofTcm A2
approximately the same from the plasmids in whichtcmG expression was under the control of either ermE* or tcmG pro- oxygenase has a M , of 61.000 as drterminrd by grl filtration
moter alone (Fig. 2A, abundance in lanes 2-4 = 1, 0.89, and chromatography on a Superose 6 HR 10/80 column.
Stoichiomctr?, of thr Trm A2 O q E r n n s r Cntnl.vrrrl l!,vdro.r.v0.89), the tandem ermE*::tcmG promoter system resulted in
Iation of 6 to I-No discrete intermrdiatr was drtrctrd in t h r
R. G. Summers. G. Meurer, and C. R. Hutchinson, unpublished data. conversion of 6 to 1 catalyzed hy Tern A2 oxygmasr undrr all
~
Tetracenomycin A2 oxygenase from S . glaucescens
30730
A.
Originally assigned TcmG>fMet-P-V-S-D-R-P-K-G-C-IS-ATCGCAGGGATGACTCGTOTaCCCGTTTCCGACCGACCG~~CT~ATC~QTCCACTG~G~GTTCCGGTACTGATAGTC~C-3’
Revised TcmG>jMet-S-T-E-E-V-P-V-L-I-VB.
1 STEEvmnIV GGGLTGLSAA
LFLSQHGVSC
RLVEIC-IRGTT
VLTRASGISS RrmELLRGvG LERTVIERGP K
L
V
E
G
A
R
W LGQPADQIPW WIRANGLHD
101
LENAVTVFEP SLDVGHLS!?T RPYWCGQDRZ, EPILRDEAVR RGARIDFNTR MEAFPADESG VTATIVDQATGEQSTVRARY
201
GHGTIGNAMS VLFKADLRDT VKGRRFVICYLPNPDEFGVL
3 0 1 WE!MSHNSARS YRSGRVFLAGDAAHVHPPAG
401
LIAAM;vRsP VRETLGITRT
QLPWAVLQ LFDFDRWIFG FFFDPRETSPEQFDERCAQIIRTATGLFG
LpvEvQMARp
AHNLSLWKLAA VLKGTAGIXL LDTYEQERLP IGAAvAlX2Ah’
IRIFIWRLNDS EELRWLLRES
TLVATGYRYT SDAVLGAAYP EPIPAAHDLT GRPGYRVPHV WLGRGGERVS !
I
’
V
D
I
C
X
W
VLAGPJXGEWQAAADWAQD
LGvpvHcHw G G W L m P D
IlFGANGGIQD
5 0 1 GAF‘STTGLT RNGALLIRPD GFVAWRAFYL PEDAAGELRS ALEFILARTS GTFGGTALEG *
FIG.3.NH,-termindportion of the tcmG gene (A) and amino acid sequence of the TcmA2
oxygenase translated
from the‘ITG start
site in tcmG ( B ) .Both the GTG and W G translational start sites are shown in boldface type; the NH, terminus determined from the purified
TcmG is shown in italic type; the putative FtBS of tcmG is underlined.
TABLE
I11
Stoichometry of the Tcrn A2 oxygenase catalyzed hydroxylationof
Tcm A2 to Tcm C
Assay description
Relative activity
%
Complete”
-TcmG
-TcmG
TcmG + boiled
-NADPH
-0, + N,6
-NADPH + NADH
100
0
0
0
0
57.3
a The complete assay solution of 250 pl with 10% (v/v) dimethyl sulfoxide consisted of 100 p 6, 250 p~ NADPH, and 1 m DTT in 50 mM
ethanolamine-HC1buffer, pH 9.0, in the presence of 5.85 pg of TcmG
was incubated at 25 “C for 10 min and then analyzed by the HPLC
method described under “Experimental Procedures.”
The assay solution was sealed with a septum stopper and evacuated
and flushed with N, several times, then 6 was introduced into the assay
solution via the septum stopper, incubated, and analyzed as described
above.
1
b
I
conditions studied. As summarized in Table 111, the enzyme
utilizes either NADH or NADPH as electron donors and
0, by exchange with
requires molecularoxygen;removing
nitrogen inhibited the hydroxylation completely, as did heat
denaturation.
pHDependence-Tcm A2 oxygenase displayed an optimal pH
of 9.0-9.5 in 50 mM Tris-HC1or 50 nm ethanolamine-HC1buffer
as shown in Fig. 4.While a decrease of 1 unit below the optimal
pH caused an approximately 50%loss of the specific activity, an
increase of 1 unit above the optimal pH resulted in complete
loss of the enzyme activity.It is not known, however, ifthe loss
of enzyme activity a t pH > 9.5 resulted from deprotonation of
specific amino acid residues at the active site, from denaturation of the protein, or from decompositionof NADPH.
Prosthetic Group Investigation-Most of the known oxygenases have either a flavin or heme as their prosthetic group or
require a metal ionfor the activation of molecular oxygen.
Since analysis of the nucleotide sequenceof tcmG has revealed
a conserved domain for flavin binding (20) and a flavin has
characteristic absorption maxima at 375 and 450 nm (361,we
determined the UV-VIS absorption spectrum of the purified
Tcrn A2 oxygenase (Fig. 5). These data show that Tcm A2 oxygenase is a flavoenzyme. To further identify the nature of this
flavin prosthetic group, a solution of TcmA2 oxygenase (224 pg)
was heat denatured to release the non-covalently boundflavin
(33),which then was analyzed by HPLC ona C,, column. Under
the given conditions, mostof the known flavin derivatives such
as FAD and FMN are well separated (341,as shown in Fig. 6A.
Fig. 6B shows that theprosthetic group released from Tcm A2
PH
FIG.4. pH dependence of the Tcm A2 oxygenase in 50 m~ TrisHCland 50 n m ethanolamine-HC1 buffer. Assay conditions and
HPLC analysis are described under “Experimental Procedures.”
oxygenase is FAD, which was further confirmed by co-chromatography with a mixture of authentic FAD and FMN (Fig. 6 0 .
From the results of HPLC analysis, calibrated with authentic
FAD, it was established that themolar ratio of apo-TcmG/FAD
is 1:l. This value agrees reasonably well with a 1:0.73ratio of
apo-TcmG/F’AD, determined spectroscopically based on the moand €!Em,respectively.
lar absorbance indexes of E”,;“
Preparation of Apo-Tcm A2 Oxygenase and Its in Vitro Reconstitution with FAD-After establishing that Tcm A2 oxygenase contains 1 molof non-covalently bound FAD as a prosthetic group, we exploredways t o prepare the apo-Tcm A2
oxygenase. The best results were obtained by dialysis of Tcm A2
oxygenase in 100 nm potassium phosphate buffer, pH 4.0, in
The protein was completely
the presence of 2 M KC1 (35, 36).
denatured by this treatment as indicated by its precipitation
and was resolubilized and presumably refolded by subsequent
dialysis in 25 nm Tris-HC1, pH 8.0, 1 mM DTT. The apoenzyme
was colorless, in contrast to the characteristic yellow color of
the holoenzyme, suggesting the removal of the flavin prosthetic
group, and possessed very little of the initial enzyme activity
(Table IV).The apo-Tcm A2 oxygenase could be
partially reconbut only by FAD; addition of either
stituted in uitro (35, 36),
FAD or FMN t o the holo-TcmA2 oxygenase under parallel
conditions resulted in a very small change in activity.
Tetracenomycin A2 oxygenase from S. glaucescens
30731
TABLE
lV
In vitro reconstitution of apo-Tcm A2 oxygenase with FAD and FMN
Assay description
Relative activity
Holoenzyme
Holoenzyme + FAD
Holoenzyme + FMN
Apoenzyme
Apoenzyme + FAD
Apoenzyme + FMN
100
115
%
Abs
"
0.0
300
400
500
23
<4
a The complete assay solution of 250 pl with 10% (vh) dimethyl sulfoxide consisted of 100 UM 6.1.0 mM NADPH, and 1 mM DTT in 50 mM
ethanolamine-HC1buffer, pH 9.0, in the presence of 5.85 pg of holo- or
apo-Tcm A2 oxygenase withlwithout FAD or FMN, respectively, was
incubated at 25 "C for 10 min and then analyzed by the HPLC method
described under "Experimental Procedures."
600
200
87
<4
600
bition of the oxygenase with P-450 inhibitors (46, 47). At least
three pathways can be proposed for their introduction. They
FIG.5. W-VIS absorbance spectrum of the Tcm A2 oxygenase could simply beretained from the carbonyl groups of polyketide
(1.12 mg/ml in 26 l l l ~Tris-HCl, pH 8.0).
precursors, such as acetate, malonate, etc., without going
through an aromatic intermediate like 6, asin the urdamycins
400 - A at 450 nm
A
(42) or 5, whose 4a-OHwas found t o be derived fromacetate (8).
Alternatively, they could be introduced late in the biosynthetic
pathway by an oxygenase, as proposed for 1 in Fig. 7, acting
200
as
either
a
monooxygenase (route a ) or a dioxygenase
FAD FMN
(route b).
The purification and characterization of Tcm A2 oxygenase
supports the hypothesis that the triple hydroxylation of 6 to 1
is catalyzed by a single enzyme. This enzyme requires molecular oxygen and is able to use eitherNADH or NADPH. Since
the apparent V',JK
for',NADPH (0.179) is more than 3-fold
larger than that for NADH (0.0565),we conclude that Tcm A2
oxygenase prefers NADPH under physiologicalconditions.
NH,-terminal amino acid analysis of the purified Tcm A2 oxyC
genase confirmed that itwas encoded by tcmG but showed that
translation begins at a rare TTG codon instead of the much
more frequently used GTG or ATG translational start site (48)
assigned initially on the basis of nucleotide sequence analysis
(Fig. 3A) (20). SDS-PAGE analysis showed that the purified
enzyme displayed a single band with M,60,000, and gel filtraa
tion chromatography suggested that thenative enzyme has an
9.0
13.0
M, 61,000, indicating that Tcm A2 oxygenase is a monomeric
Time (min)
FIG.6. HPLC analysis of FAD prosthetic group dissociated protein in solution.
The exact mechanism of the Tcm A2 oxygenase catalyzed
from the Tcm A2 oxygenase. A, a mixture of authentic FAD and
FMN; B , the flavin prosthetic group dissociated from the Tcm A2 oxy- hydroxylation of 6 to 1 is not clear yet since the data reported
genase; C , the dissociated flavin prosthetic group was co-chromatogra- here do not discriminate between a monooxygenase and a diphied with the mixture of authentic FAD and FMN analyzed in A.
oxygenase mechanism. As proposed in Fig. 7 (route a ) , two of
the threeoxygens could beintroduced stepwise from molecular
Kinetics-Assuming that the 0, concentration was constant oxygenif the enzyme acts like a monooxygenase. The first
in the assay solution, kinetic analyses were carried out on the monooxygenase activity could hydroxylate 6 to hydroquinone 7
basis of a pseudo-first-order treatment with a steady-state
that could be further oxidized by the second monooxygenase
approach. Thus, the effect of the initial concentration of 6 activity t o yield epoxyquinone8; cis opening of oxirane ring by
on the formation of 1 was determined at the concentration of a H,O molecule could introduce the third oxygen to yield dihyNADPH 2 10 K2mpH,and the effect of NADH or NADPH was droxyquinone 9 that could be finally reduced to 1.In contrast,
Velocities two of the threeoxygens could also be
determined at the concentration of 6 2 10 KEmA2.
introduced in aconcerted
were then fitted to the Michaelis-Menten equation (32) and the fashion from molecular oxygen if the enzyme acts as a dioxy'
,for 6,NADH, and NADPH were found to be 1.81 genase (Fig. 7,route b ) where a likely stableintermediate
apparent K
'' 0.38, 260 -c 19, and 82.1 2 17 p ~ respectively,
,
with an ap- would be the epoxysemiquinone 10;cis opening of its oxirane
parent V m m of 14.7 f 1.1nmol Tcm C/min-mg.
ring by a H,O molecule could introduce the third oxygen to
yield 1. Both mechanisms are consistent with the results of an
DISCUSSION
feeding experiment that hasdemonstrated that the
in uiuo 1802
The enzymatic mechanism for the introduction of angular oxygens of the 4-OH and 12a-OH groups come from molecular
hydroxy groups like 4a-OH and 12a-OH of 1 into many other oxygen and the 4a-OH group presumably comes from H,O (6).
naphthacenequinone, angucycline, and anthracycline antibiot- The monooxygenase pathway (Fig. 7, route a ) is supported by
ics (4-8, 42) is unknown, and the origins of such groups have the amino acid sequence similarity between TcmG (20) and
been studied previously only by in vivo feeding experiments other bacterial hydroxylases, such as those found in the oxyor "0-containing precursors (6, 8, 43-45) or by inhi- tetracycline producer Streptomyces rimosus (49) and the
with
Wavelength (nm)
~
30732
Tetracenomycin A2 oxygenase from S. glaucescens
OH 0
C
H
~ COZCH3
O ~ ~
OH CH3
0 OH
OH 0
OH
C
H
0
-c
H
~
3
O
0 CH,
0
~ O
OH
C COzCH3
H 3
CH3O*XH3
OH CH3
0
a
7
H
c3
0
*
-
J COzCH3
~ O C
0
9
C02CH3
CH,
c
H
3
0
*
m
H
3
C02CH3
6
1
I
t
OH
6CH,6
OH
6
OH CH,
u
0
u
0
OH CH3
10
FIG.7. Proposed mechanism for the Tcm A2 oxygenase-catalyzedhydroxylation of Tcm A2 to Tcm C involving a monooxygenase
(route a)or a dioxygenase (route b ) .
daunorubicin producer Streptomyces peucetius (50) that acton
tetracyclic aromatic substrates similar to 6.Monooxygenases
that oxidize hydroquinone to form epoxyquinones also have
been isolated recently from Streptomyces LL-C10037 (51) and
Streptomyces MPP 3051 (51),and a monooxygenase activity for
the direct oxidation of an anthraquinone to an epoxyanthraquinone has been reported inStreptomyces rosa var. notoensis
OS-3966 (45). Moreover, 7 has been isolated as a stable metabolite by refluxing an acidic solution of 1 (521, and a similar
structure like 9, in fact, has been isolated from Streptomyces
olivaceus TU 2353 (4, 5 ) , the producer of 2, as the minor meif elloramycin E
tabolite elloramycin E. (It was not established
is the directprecursor of 2 or resultsfrom a facile oxidation of
2.) The dioxygenase pathway (Fig. 7, route b ) is consistent with
our inability to detect any discrete intermediate in thein vitro
hydroxylation of 6 to 1 (10might be spontaneously hydrolyzed
to 1 upon release from the enzyme). We favor the dioxygenase
mechanism since in thiscase the monomeric Tcm A2 oxygenase
would only need to recognize one substrate, 6,instead of recognizing at least three substrates, 6,7,and 9, as in the monooxygenase mechanism, assuming that theconversion of 8 to
9 is also a spontaneous process. Furthermore, a dioxygenase
mechanism similar to route
b has recently been established for
the vitamin K-dependent carboxylase (53, 541, although the
latter enzyme does not possess any prosthetic group or require
NAD(P)H cofactors.
Many bacterial oxygenases are flavoenzymes, and FAD and
FMN are themost common forms of the flavin prosthetic group
identified (36). It is known, however, that unusual flavin derivatives are utilized as cofactors in Streptomyces spp. For instance, a 5-deazaflavin is required for the anhydrotetracycline
oxygenase from S. rimosus (491, and an unknownflavin is
implicated in a tylosin reductase from Streptomyces frudiae
( 5 5 ) .Tcm A2 oxygenase contains 1mol of FADthat binds to the
apoenzyme non-covalently, as judged by its release from the
protein upon heat denaturation (33). These facts led to the
preparation of apo-Tcm A2 oxygenase and its attemptedreconstitution invitro. Apparentlythe apoenzyme is much less
stable than theholoenzyme, yet the holoenzyme can be reconstituted by FAD only, albeit to a low degree (Table IV). The
latter observation re-enforces the conclusion that Tcm A2 oxygroup. It is
genaseuses FAD exclusively asitsprosthetic
known that upon removal of the flavin prosthetic group from
some flavoenzymes the apoenzyme becomes less stable (35,36,
561, as observed here with the apo-Tcm A2 oxygenase, which
failed to be reconstituted completely after being kept at -20 "C
for 3 days. Although it is not clear why exogeneous FAD or
FMN slightly activates or inhibits the enzyme (Table IV), the
inhibitory effect of exogeneous flavins has been reported for
other flavoenzymes and interpreted as resulting from competitionbetween the freeflavin and enzyme-bound flavin for
NAD(P)H as electron donor (55). On the other hand, it isalso
known that a non-covalently bound flavin prosthetic group can
be lost in the course of protein purification (57, 58), leading t o
a fortuitous activation of the enzyme preparation when it is
supplemented by externally added flavin.
It is interesting to
point out thedifference between the single
and double promoter systems in theefficiency of expression of
tcmG. The revised tcmG translational start site, unfortunately
discovered after the expression vectors were made, abolished
the influence of the two different RBSs in these constructs
since
the GAAAGGCTGC RBS that precedes the TTG translational
start sitewas presumably utilized in all constructs. Therefore,
since the expression of tcmG under thecontrol of either tcmG or
ermE* promoter alonegave an approximately equal abundance
of TcmG (Fig. 2A, lanes 2 4 ) whereas the tandem
ermE*::tcmG
promoters resulted inapproximately 5-fold higher level of tcmG
expression (Fig. 2A, lane 11, the two promoters placed in tandem have an additive effect on expression. Similar effects of
dual promoters on gene expression have been seen in other
cases (59).
Acknowledgments-We thankKrishna
Madduri, Guido Meurer,
Evelyn Wendt-Pienkowski, Bruce Jarvis, Mark Gallo, Sharee Otten,
and Heinrich Decker for advice and discussions during thecourse of this
work and JaneWalent and Ronald Niece for the NH,-terminal sequence
analysis of tcmG.
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