Activity Assays ADPG Synthesis Direct Assay This assay is used to measure the rate of conversion of glucose-1-phosphate (G1P) and ATP to ADP-glucose (ADPG) and inorganic pyrophosphate (PPi). The substrate G1P, labeled with 14C, is converted to [14C] ADPG PPase which is subsequently separated from excess substrate via binding to positively charged DE-81 filter discs and quantified via liquid scintillation counting (LSC). Post assay, the left over G1P is digested by alkaline phosphatase allowing for the uncharged labeled glucose to be washed from the filter. This assay is used to determine physiologically-relevant kinetics of the purified enzyme. Pyrophosphorolysis Assay This assay allows for the measurement of activity in the physiologically “reverse direction” conversion of ADPG and PPi to G1P and ATP. The formation of product, [32P] ATP, was measured by quantifying the amount of [32P] ATP (with 32P phosphate released via acid hydrolysis) after separation by binding to activated charcoal using LSC. Under our conditions, the assay is specific and accurate during purification and avoids the potential for randomization of the label if the synthesis assay was used.

Starch is a complex carbohydrate essential to plants and bacteria as an energy storage compound.

The rate-limiting step in starch biosynthesis is catalyzed by the allosteric enzyme ADPGlucose

pyrophosphorylase (ADPG PPase) thereby making it an attractive candidate for protein engineering to

produce a higher yield of of the biodegradable and renewable carbon source starch. In Agrobacterium

tumefaciens (Ag.t), ADPG PPase is activated by fructose-6-phosphate (F6P) and pyruvate and is

inhibited by phosphate and sulfate. Previous molecular models of ADPG PPase have indicated that the

enzyme consists of a homotetramer structure composed of an N and C terminal connected by a loop

region. This loop region, including P288, had been determined to be important in allosteric regulation of

the E. coli enzyme. In addition, x-ray crystal structure of the Ag.t enzyme indicates that E304 is part of

an allosteric site that is comprised of residues from both the N and C terminus. In order to test these

hypotheses, the P288D and E304A and E304D enzymes were generated by site-directed mutagenesis

and expressed and purified for kinetic and physical studies. Confirming previous work, the P288D

enzyme was shown to exhibit high activity in the absence of activator and was relatively insensitive to

both activators and inhibitors. Large scale pure preparations of P288D have been prepared via phenyl

sepharose, anion exchange, and Cibacron Blue chromatography for future crystallization trials. A total of

2.4 mg (1.63 mg/mL) of pure P288D enzyme was obtained. Activator saturation plots were performed

for the E304A and E304D enzymes; the A0.5 values for fructose-6-phosphate (F6P) and pyruvate were

found to be 8.1 µM (fold activation 4.18) and 10 µM (4.24 fold activation), respectively, for the E304A

enzyme and 43.5 µM (fold-activation 3.69) and 170.2 µM (fold-activation 2.66), respectively, for the

E304D enzyme. In addition, the E304D enzyme was activated by FBP (A0.5 = 0.40 mM (fold-activation

2.34). The wild-type enzyme displays A0.5 values ~10 fold higher than the E304A enzyme and fold

activation for F6P and pyruvate 3 and 2 fold higher than E304A, respectively. The A0.5 values for E304D

were more similar to wild-type while the fold-activation for F6P and pyruvate were ~6-fold and ~3-fold

lower than wild-type. The results indicate that both the long loop structure and the site that includes

E304 are important for regulation. Grant number is P031C110116.

Small scale or large scale growth and

induction in EA345 E. coli cells

Cell and Protein Harvesting

ADPG PPase Purification

Kinetic Characterization

Emmanuel Silva1, Hoomai Karzai1, Vonice Benjamin1 ,Andrew Orry2, and C. R. Meyer1 1Chemistry and Biochemistry Department at California State University, Fullerton and 2Molsoft LCC, La Jolla, CA

Uno Q6

(Anion Exchange Column)

Phenyl Sepharose

(Hydrophobic Column)

(NH4)2SO4 Precipitation

(30% Saturation)

Heat Step

(5 minutes at 65˚C)

Blue A Column

(Affinity Column)

Organism Activators Inhibitors .

E. coli FBP AMP, ADP

Rs. r. Pyruvate None

Ag. t. F6P, Pyruvate Pi, ADP, AMP, Sulfate

Rb. s. F6P, FBP, Pyruvate AMP, ADP, Pi, PEP

T. ma. FBP -

Plants 3PGA Pi

Regulation of ADPG PPase

Table 1. Allosteric regulators of ADPG PPase vary by

organism.

• Starch (in plants and algae) and glycogen (in bacteria) are sources of renewable carbon and energy

• Improving starch synthesis efficiency is important for various industries:

• Food - complex carbohydrate in plant crops (ex. rice, wheat and potatoes)

• Pharmaceuticals, cosmetics, packaging, plastics, and bioethanol – complex and natural starch

• Studying the enzymes critical in starch biosynthesis will allow for protein engineering to

develop specialty starches and to improve starch production efficiency

• ADP-Glucose pyrophosphorylase (ADPG PPase):

• Produced from glgC gene

• Converts glucose-1-P (G1P) and adenosine triphosphate (ATP) to ADP-glucose and

pyrophosphate (PPi)

• Catalyzes the rate-limiting step of the three-step starch biosynthesis pathway (M. A. Ballicora et

al., 2004) (Fig. 1)

• An attractive target for protein engineering to increase the production of renewable carbon

• Regulated differently in different organisms (Fig. 2)

Figure 2. Ag. t. ADPG PPase structure and allosteric site molecular modeling (Cupp-Vickery et al., 2008) depicting (a) a ribbon diagram of

enzyme with space-filling models of sulfate, ATP, G-1-P, (b) the sulfate-binding site, (c) a space-filling model of enzyme with F-6-P (yellow) in

proposed activator-binding site, ATP (orange) and G-1-P (purple) in active site, and basic (blue), acidic (red), and polar (green) residues, and (d) the

proposed F-6-P binding site and possible participating residues. (S: yellow, O: red, C: green, N:blue, P: brown)

ADPG PPase Structure

Starch Biosynthesis Pathway

Glucose-1-phosphate ADP glucose

ADP glucose (glucosyl)4

(glucosyl)5

starch synthase

ADP-glucose pyrophosphorylase

branching enzyme

Figure 1. ADP-glucose pyrophosphorylase (ADPG

PPase) catalyzes the rate-limiting step of starch

biosynthesis in plants and glycogen biosynthesis in

bacteria. ADPG PPase converts glucose-1-phosphate

(G1P) and ATP to ADP- glucose and PPi (upper left).

The activated form of glucose is then added to a

growing glycan chain by starch synthase by a α 1,4

linkage (lower left). The glycan macromolecule

becomes more tightly packed as branching enzymes

reattaches fragments of long, growing glucosyl chains

as branches of other chains (α 1,6 linkages, below).

S0.5(mM) Vmax (U/mg)

ATP Hill #

No Effector 0.21 ±0.04 1.3 ± 0.2 12.1 ±1.0

WT + 2 mM F6P 0.051 ±0.004 1.3 ±0.1 147.9 ±4.1

+ 2 mM Pyruvate 0.10 ±0.01 1.9 ±0.03 105 ±5

No Effector 0.55 ± 0.03 2.9 ± 0.3 52.2 ± 1.9

P288D + 2 mM F6P 0.40 ±0.04 1.4 ± 0.1 59.5 ± 3.2

+ 2 mM Pyruvate 0.45 ± 0.02 2.3 ± 0.3 54.9 ± 1.7

Verification Gel Kinetics Data for P288D Ag.t ADPG PPase

Figure 3. SDS-PAGE of samples P288D mutant purification: 10%

Resolving Gel, 5% Stacking Gel, Electrode Buffer (0.025 M Tris,

0.192 M Glycine, 1% SDS, pH 8.3), 1.5 hours, 100 V, M –

Molecular Weight Marker, 1 – crude extract (~15 µg), 2 – Heat

step/(NH4)2SO4 (~13 µg), 3 – Phenyl Sepharose (~6 µg), 4 – Uno

Q (~2µg), 5 – Blue A (~2 µg).

Table 2. Kinetic Data for WT, P288D and P288E Ag. t. ADPG PPase including S0.5 (mM) and

Hill numbers for the substrates ATP, Vmax (U/mg) values are shown from ATP saturation. The

data was determined using ADPG synthesis direction assays; the assays were performed in

presence of 100 mM HEPES pH 8.0, 0.5 mg/ml BSA, and saturating concentrations of the non-

varied substrate or co-factor (i.e. 2 mM ATP, 10 mM MgCl2, and 0.5 [14C] glucose-1-

phosphate).

M 1 2 3 4 5 6

22

97 kDa

66

45

31

50 kDa

•Previous molecular and kinetic data for P288D showed desensitization to both

inhibitor and activators; however, in the presence of no activators P288D was

determined to have a ~2.5 fold increase in S0.5 values for ATP when compared to

the wild type resulting in a ~4 fold increase in Vmax.

•In order to determine-function relationships, P288D was successful expressed

and purified to near homogeneity in preparation for future crystallization trials and

size exclusion chromatography

•Kinetic analysis of purified E304D enzyme showed:

oActivation by Fructose-1,6-biphosphate, although WT is insensitive (A0.5

=0.40mM, 2.34 fold-activation)

oExhibited A0.5 values for fructose-6-phophate (F6P) and pyruvate similar to

WT

oExhibited ~6 fold and ~3 fold activation lower than WT for F6P and

pyruvate respectively

•Kinetic analysis of purified E304A enzyme showed:

oExhibited 8.1 µM (fold activation 4.18) and 10 µM (4.24 fold activation) A0.5­

values for F6P and pyruvate respectively

oExhibited A0.5 values ~10 fold lower than WT indicating higher apparent

affinity

oExhibited fold activation 3 and 2 fold lower then WT for F6P and pyruvate

respectively

•Crystallization trials of the P288D altered protein as a first step toward

solving the three-dimensional structure for comparison to WT

•Size-exclusion chromatography for further purification and determination of

the aggregation state of P288D and other Ag.t. altered proteins for

comparison to WT

•Generation of different mutations derived from the loop region for further

investigation of allosteric regulation mechanism

•Truncation, shortening the loop region, to determine structure-function of

ADPG PPase and importance of the loop region in allosteric regulation

Meyer Lab Team

Department of Chemistry and Biochemistry,CSUF

STEM2 (Supported by the Department of Education)

A0.5(µM)

F6P Hill #

Fold

Activation Pyr. Hill #

Fold

activation FBP Hill #

WT 130 ± 10 2.0± 0.3 12 100 ± 10 1.9 ± 0.3 8.5 N/A N/A N/A

E304A 8.9 ± 1.1 1 4.18 90.0 ± 2.3 1 3.24 N/A N/A N/A

E304D 43.5 ± 19.7 1 3.69 170.2 ± 45.5 1 2.66 402.8 ± 38.9 2 2.34

Kinetics Data for E304A and E304D Ag.t ADPG PPase

Fold

Activation

0 100 200 300 400 500

Activator (æM)

0

4

8

12

Fold

-Act

ivat

ion

WT+F6P

+Pyr

0 50 100 150

Activator (æM)

0

1

2

3

4

Fold

-Act

ivat

ion

E304D+F6P

+Pyr

0 100 200 300 400 500

Activator (æM)

0

2

4

6

Fold

-Act

ivat

ion

E304A

+F6P

+Pyr

Activator

(mM)

Activator

(mM)

Activator

(µM) Activator

(µM) Activator

(µM)

Fo

ld a

ctivatio

n

Fo

ld a

ctivatio

n

Fo

ld a

ctivatio

n

Activator Saturation Plots for E04A and E304D Ag.t ADPG PPase

Table 3. Kinetic Data for WT, E304A, and E304D Ag.t ADPG PPase including A0.5 (µM) and Hill numbers for the activators F6P, Pyr., and FBP and fold activation

values are shown. The data was determined using ADPG synthesis direction assays; the assays were performed in presence of 500 mM HEPES pH 8.0, 10 mg/ml

BSA, and saturating concentrations of the non-varied substrate or co-factor (i.e. 50 mM ATP, 200 mM MgCl2, and 11.8 [14C] glucose-1-phosphate).

Figure 4. Activator saturation plots of WT and E304A and E30D Ag.t ADPG PPase depicting folding

activation vs. activator concentration (µM). The assays were performed in presence of 500 mM HEPES

pH 8.0, 10 mg/ml BSA, and saturating concentrations of the non-varied substrate or co-factor (i.e. 50 mM

ATP, 200 mM MgCl2, and 11.8 [14C] glucose-1-phosphate).

Meyer, Christopher, Jennifer Yirsa, Bruce Gott, and Jack Preiss. 1998. A Kinetic Study of

Site-directed Mutants of Escherichia Coli ADP-glucose Pyrophosphorylase: The Role of

Residue 295 in Allosteric Regulation. Archives of Biochemistry and Biophysics. 352, no.

2: 247-254.

Gomez-Casati, Diego, Robert Igarashi, Christopher Berger, Mark Brandt, Alberto Iglesias,

and Christopher Meyer. 2001. Identification of Functionally Important Amino-terminal

Arginines of Agrobacterium Tumefaciens ADP-glucose Pyrophosphorylase by Alanine

Scanning Mutagenesis. Biochemistry. 40, no. 34: 10169-10178.

Cupp-Vickery, Jill, Robert Igarashi, Marco Perez, Myesha Poland, and Christopher Meyer.

2008. Structural Analysis of ADP-glucose Pyrophosphorylase from the Bacterium

Agrobacterium Tumefaciens. Biochemistry. 47, no. 15: 4439-4451.

Ballicora, Miguel, Alberto Iglesias, and Jack Preiss. 2004. ADP-glucose

Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis. Photosynthesis

Research. 79, no. 1: 1-24.