Purification of Pyrophosphate-Dependent
Phosphofructo-1-kinase of Borrelia burgdorferi
Amber Pentoney, Aytug Tuncel and Thomas W. Okita
Interdisciplinary Biochemistry
METHODS
ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme
in glycogen synthesis in bacteria and starch synthesis in higher plants.
The enzyme uses glucose 1-phosphate (G1P) and ATP as substrates to
produce ADPglucose (the activated form of glucose for the next
biosynthetic enzyme) and inorganic pyrophosphate (PPi) (Fig. 1). In
general, the enzymatic activity assay of this reaction is performed by
measuring the amount of 14C-labeled ADPglucose produced from 14Clabeled G1P and ATP. This method, however, has the disadvantage of
working with radioactive material and its accompanying restrictive
regulations in usage and record keeping. Moreover, it is relatively time
consuming. Alternatively, the assay can be performed enzymatically by
measuring the amount of PPi produced by AGPase. This can be
accomplished using PPi-dependent phosphofructokinase (PPi-PFK),
aldolase, triosephosphate isomerase and glycerol-3-phosphate
dehydrogenase (NAD+) (Fig. 2) and then measuring the conversion of
NADH to NAD+ by measuring the decrease in absorbance at 340 nm.
To perform the spectroscopic assay it is essential to obtain purified
PPi-PFK, the only enzyme in this scheme which is not available
commercially. Therefore, I purified PPi-PFK from Borrelia burgdorferi by
expressing it in Escherichia coli BL21 cells and applying a two-step
purification protocol. First, the bacterial crude extracts were treated
with polyethyleneimine to remove DNA followed by Sephadex G-25
size exclusion chromatography. The nucleic acid free extracts were
then fractionated on cellulose phosphate bi-functional cation exchange
chromatography. Using this protocol I was able to purify the protein
to near homogeneity and assess its kinetic properties.
G1P + ATP  ADPGlc + PPi
FIGURE 1. AGPase catalyzes the rate-limiting step of the starch biosynthesis
pathway.
INTRODUCTION
Starch is a main reserve stored in many harvestable edible sink organs of
plants and, hence, a major constituent of the human diet. Understanding
the biosynthesis of starch can help identify ways to increase starch yield
and, in turn, increase overall plant productivity and crop yield, processes
dependent on source-sink relationships. Source-sink relationship can be
described as the extent to which a plant can photosynthesize and fix CO2
through source leaves, and the extent to which the fixed carbon can then
be assimilated by sink tissues to form reserves such as starch (1). Enzymes
involved in the overall pathway can be manipulated to obtain a higher yield
of starch production. ADP-glucose pyrophosphorylase (AGPase) is an
important enzyme that catalyzes a rate-limiting step of starch biosynthesis
(2). When AGPase is available, it catalyzes the reaction of glucose 1phosphate (G1P) and ATP to produce ADPglucose and inorganic
pyrophosphate (PPi). Assays of the reaction are normally monitored with
radiolabeled 14C-G1P. In order to continue our studies without using
radioisotopes, we have developed an alternative assay through coupling
additional enzyme reactions (Fig. 2). To proceed, it was essential to purify
PPi-dependent phosphofructokinase (PPi-PFK), the only enzyme
commercially unavailable. TABLE 1 shows the overall purification process
where thirty-six percent of PPi-PFK is purified and recovered from crude
extract.
PPi-PFK Assay.
All activity assays of PPi-PFK were performed at room temperature using a
spectrophotometer to measure the decrease in absorbance at 340nm until no further
changes in absorbance were observed. Assays were performed in 100 µl of solution
containing 20 mM KTes (pH 7.2), 1 mM EDTA, 5 mM MgCl2, 2 mM F6P, 0.2 mM NADH, and
0.25 units each of aldolase, triosephosphate isomerase, and glycerol-3-phosphate
dehydrogenase.
AGPase Assay
AGPase was assayed in a 200 µl solution of 100 mM Hepes (pH 7.0), 3 mM DTT, 10 mM
MgCl2, 2 mM ATP, 5 mM 3-PGA, 2 mM G1P and 0.4 mg/ml BSA. Assays were initiated by
addition of enzyme and performed at 37°C for 2-6 minutes. Reactions were then stopped
by boiling the samples for one minute. The amount of PPi produced by AGPase was
determined from a standard curve (Plotted by different concentrations of NaPPi and
omitting the enzyme) and measured spectrophotometrically at 340nm by mixing 100 µl of
the first reaction and 200 µl of the developing mixture. The developing mixture contains
final concentrations of 25 mM imadazole (pH 7.2), 1 mM EDTA, 5 mM MgCl2, 0.2 mM
NADH, 2 mM F6P, and 0.4 units each of aldolase, triosephosphate isomerase, glycerol-3phosphate dehydrogenase and 0.8 µg of the purified PPi-PFK. Both solutions were
prepared
daily.
Sephadex-G25
kDa
170
130
T0 T20 CE
4 6
8 10
12
TABLE 1. Volume represents the entire volume of the material specified. Protein concentrations were measured at ABS280/260.
Recovery shows the amount of protein obtained from initial crude extract. Our crude extract used was obtained after PEI and
centrifugation.
Purif
60.00
50.00
Specific Activity
(µmoles/min/mg)
ABSTRACT
Expression and purification of recombinant PPi-PFK.
To purify PPi-PFK the methods used were in accordance with Deng et al (3). The plasmid
encoding the enzyme was first transformed into E. coli BL21 (DE3) cells, plated onto
ampicillin (200μg/ml) media and incubated overnight. Three colonies were then incubated
into 25 ml of LB media and grown overnight at 37 °C. The seed culture was then
transferred into one-liter of NZCYM media (10 g NZ-amine, 5 g yeast extract, 5 g NaCl, 2
g MgSO4 ∙ 7 H2O and 1 g casamino acids, pH 7.0) and incubated at room temperature
until optical density (OD600) reached ~0.7. The cells were induced with a final
concentration of 0.1 mM IPTG for twenty hours. The cells were centrifuged and resuspended in 25ml of binding buffer (20 mM Pipes pH 7.0, 0.1 mM EDTA, 1 mM DTT). The
cells were then disrupted by sonication and the cell debris pelleted by centrifugation at
20,853g for 20 minutes. The supernatant fluid was collected and 10% polyethyleneimine
(PEI) was slowly added to a final concentration of 0.1% and mixed for ten minutes. The
precipitated nucleic acids were then removed by centrifugation at 10,000g for 10 minutes.
The resulting supernatant was then passed through Sephadex-G25 column (15cm x 2.5cm)
to remove excess PEI. The fractions were run on a 12% polyacrylamide gel, and fractions
containing the enzyme were combined. These combined fractions were then passed
through a phosphocellulose (P11, Whatman) column (5cm x 2.5cm). After extensive
washing, the enzyme was eluted with binding buffer containing 0.5mM fructose 1,6
bisphosphate. Fractions from both columns were tracked by analysis of samples on SDS
polyacrylamide gels and assayed using a spectrophotometer at 340nm. Fractions containing
PPi-PFK were combined, made to 50% glycerol and stored in a -20°C freezer. The enzyme
was then utilized to test the alternative assay performed in the absence of radioisotopes.
40.00
Radioactive
Non-Radioactive
30.00
20.00
10.00
0.00
SWTLWT
SSiLRK
SSiLRKA
FIGURE 4. Preliminary data comparing the potato AGPase activity measured
by radioactive and non-radioactive assay methods. SWTLWT is a wildtype
AGPase while SSiLRK and SsiLRKA are two mutant AGPase enzymes. SSi denotes a
D143N mutation in the small subunit, LRK denotes mutations at K41R and
T51K in the large subunit, while LRKA contains K41R, T51K, and S155A
mutations in the large subunit.
CONCLUSIONS
Using a combination of PEI precipitation and, Sephadex G-25 and P11
column chromatography steps, PPi-PFK was successfully purified to near
homogeneity. In FIGURE 3B a single polypeptide band corresponding to
PPi-PFK can be seen clearly at 62kDa in fractions eight through eleven. PPiPFK was used in preliminary tests for an alternative assay not requiring the
use of radioactive substrates. FIGURE 4 shows a direct comparison of the
enzyme activities measured by radioactive and non-radioactive assays.
Non-radioactive assay exhibits reliability, and the assay will continue to be
optimized. The enzyme can be utilized on additional mutants in the
alternate assay.
14 16 18 20 22
95
REFERENCES
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1. Lee SK, Hwang SK, Han M, Eom JS, Kang HG, Han Y, Choi SB, Cho MH, Bhoo SH, An G, Hahn
TR, Okita TW, Jeon JS. (2007) Identification of the ADP-glucose pyrophosphorylase isoforms
essential for starch synthesis in the leaf and seed endosperm of rice (Oryza sativa L.). Plant
Molecular Biology 65, 531-546.
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2. Preiss J, Sivak M.
(1996) Starch synthesis in sinks and sources. In E Zamski, ed, Photoassimilate Distribution in
Plants and Crops: Source-Sink Relationships. Marcel Dekker, New York, pp 139-168.
26
FIGURE 3A. Analysis of enzyme fractions by 12% SDS-polacrylamide gel electrophoresis (SDS-PAGE) followed by staining with coomassie brilliant
blue. PPi-PFK gene product is shown by the arrow at 62-kDa. Lane T0 shows transformed E. coli cells before induction, T20 shows cells 20 hours
after induction, CE shows crude extract before purification, PEI shows supernatant after treatment with polyethyleneimine, PEI Precip. shows the
precipitate after PEI is added and centrifuged, Fractions 4 to 22 from the Sephadex-G25 column are shown.
3. Zhihong Deng, David Roberts, Xiaojun Wang, and Robert G. Kemp.
(1999) Expression, Characterization, and Crystallization of the Pyrophosphate-Dependent
Phosphofructo-1-kinase of Borrelia burgdorferi. Biochemistry and Biophysics 371, 326-331.
Phosphocellulose
kDa
170
130
5
6
7
8
9
10
11
12
13
14
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Aknowledgements
Dr.Tom Okita
55
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Aytug Tuncel
26
Okita Lab Members
FIGURE 2. #1-5: Reactions coupled for assay.
Enzymes:
AGPase: ADPglucose pyrophosphorylase
PPi – PFK: Pyrophosphate dependent phosphofructokinase
Aldolase
Triosephosphate isomerase
G3P:Glycerol-3-phosphate Dehydrogenase
Substrates/Products:
DHAP: dihydroxyacetone phosphate
FBP: Fructose_1,6_ biphoshphate
F6P: Fructose_6_phosphate
PPi : Inorganic pyrophosphate
NAD+/NADH: Nicotinamide adenine dinucleotide
Dr. Christopher Meyer
FIGURE 3B. Analysis of enzyme fractions by 12% SDS-PAGE. The purified PPi-PFK eluted from phosphocellulose column are shown by
the arrow at 62-kDa. Lane CE shows crude extract before purification, PEI shows supernatant after polyethyleneimine added and
centrifuged, PEI Precip. shows the precipitate after PEI is added and centrifuged, S G25 shows combined fractions from Sephadex-G25
column, p11 F.T. shows flow through, P11 Wash shows column rinsed with binding buffer, 5-14 show fractions from elution.
This work was supported by the National Science
Foundation Plant Genome Grant DBI-0605016