Information on EC 1.1.1.95 - phosphoglycerate dehydrogenase and Organism(s) Escherichia coli and UniProt Accession P0A9T0

2-oxoglutarate + NADH + H+

?

-

-

?

2-oxoglutarate + NADH + H+

D-2-hydroxyglutarate + NAD+

-

-

?

3-phospho-D-glycerate + NAD+

3-phosphohydroxypyruvate + NADH

6 entries

3-phospho-D-glycerate + NAD+

3-phosphonooxypyruvate + NADH + H+

-

-

?

3-phospho-D-glycerate + NAD+

3-phosphooxypyruvate + NADH + H+

3-phosphooxypyruvate + NADH + H+

3-phospho-D-glycerate + NAD+

-

-

r

alpha-ketoglutarate + NADH

2-hydroxyglutaric acid + NAD+

-

-

?

2-oxoglutarate + NADH + H+

D-2-hydroxyglutarate + NAD+

-

-

?

3-phospho-D-glycerate + NAD+

3-phosphohydroxypyruvate + NADH

-

287525, 287530

-

?

3-phospho-D-glycerate + NAD+

3-phosphonooxypyruvate + NADH + H+

-

738070

-

?

3-phospho-D-glycerate + NAD+

3-phosphooxypyruvate + NADH + H+

3-phosphoglycerate + NAD+

3-phosphohydroxypyruvate + NADH

13 entries

3-phosphooxypyruvate + NADH + H+

3-phospho-D-glycerate + NAD+

-

-

r

alpha-ketoglutarate + NADH

2-hydroxyglutaric acid + NAD+

3 entries

3-phospho-D-glycerate + NAD+

3-phosphohydroxypyruvate + NADH

3 entries

3-phospho-D-glycerate + NAD+

3-phosphonooxypyruvate + NADH + H+

-

-

?

3-phospho-D-glycerate + NAD+

3-phosphooxypyruvate + NADH + H+

first step of L-serine biosynthesis

-

r

3-phospho-D-glycerate + NAD+

3-phosphonooxypyruvate + NADH + H+

-

738070

-

?

3-phospho-D-glycerate + NAD+

3-phosphooxypyruvate + NADH + H+

enzyme in the L-serine biosynthetic pathway

-

?

3-phosphoglycerate + NAD+

3-phosphohydroxypyruvate + NADH

NAD+

5 entries

NADH

3-acetylpyridine-NADH

-

40% as effective as NADH

287525, 287530

deamino-NADH

-

as effective as NADH

NAD+

NADH

NADPH

-

8% as effective as NADH

2-methyl-N-(2-[[(2E)-2-[2-[(2-nitrobenzyl)oxy]benzylidene]hydrazinyl]carbonyl]phenyl)benzamide

-

4-(5-[(Z)-[1-(3,4-dimethylphenyl)-3,5-dioxopyrazolidin-4-ylidene]methyl]furan-2-yl)-N-(1,3-thiazol-2-yl)benzenesulfonamide

-

4-[(3,5-dioxo-1,2,6-thiadiazinan-4-ylidene)methyl]phenyl 2,3-diphenylquinoxaline-6-carboxylate

-

L-serine

(R)-2-amino-1-propanol

-

slightly

3-phosphoglycerate

-

Ag+

-

inhibition can be overcome by addition of dithiothreitol

amino acids

-

287525, 287547, 287548

beta-Alanine

-

slightly

glycine

hydroxyglutarate

-

product inhibition of the alpha-ketoglutarate reduction

iodoacetate

-

L-alanine

L-allothreonine

-

L-homoserine

-

L-Ser

-

binding of the inhibitor to the apoenzyme displays positive cooperativity in the binding of the first two serine molecules and negative cooperativity in the binding of the last two serine molecules. At least two NADH-induced conformational forms of the enzyme bind the inhibitor in the physiological range. Successive binding of NADH to the enzyme results in an increase in the affinity for the first inhibitor ligand bound and a lessening of both the positive and negative cooperativity of inhibitor binding

L-serine

L-threonine

-

Mercurials

-

inhibition can be overcome by addition of dithiothreitol

-

N-ethylmaleimide

-

NADH

-

inhibition of phosphoglycerate oxidation

additional information

-

0.45 - 5

2-oxoglutarate

0.19 - 320

3-phospho-D-glycerate

37 entries

0.068 - 0.081

3-phosphooxypyruvate

0.088

2-oxoglutarate

pH and temperature not specified in the publication

1.2

3-phospho-D-glycerate

pH and temperature not specified in the publication

0.0013 - 0.0032

3-phosphohydroxypyruvate

0.0032

3-phosphooxypyruvate

pH and temperature not specified in the publication

0.038 - 0.088

alpha-ketoglutarate

0.4

D-2-hydroxyglutarate

pH and temperature not specified in the publication

1.1

D-3-phosphoglycerate

0.0078 - 0.008

NAD+

0.49 - 2.1

2-oxoglutarate

3.2 - 18.7

3-phospho-D-glycerate

38 entries

3.1 - 9.6

3-phosphooxypyruvate

12

2-oxoglutarate

pH and temperature not specified in the publication, 12-33/s

0.6

3-phospho-D-glycerate

pH and temperature not specified in the publication

0.463

3-phosphohydroxypyruvate

-

apparent

2 - 8

3-phosphooxypyruvate

pH and temperature not specified in the publication

0.128 - 0.555

alpha-ketoglutarate

0.7

D-2-hydroxyglutarate

pH and temperature not specified in the publication

additional information

-

-

0.98 - 4.7

2-oxoglutarate

46 - 120

3-phosphooxypyruvate

400

2-oxoglutarate

pH and temperature not specified in the publication

0.5

3-phospho-D-glycerate

pH and temperature not specified in the publication

9000

3-phosphooxypyruvate

pH and temperature not specified in the publication

2

D-2-hydroxyglutarate

pH and temperature not specified in the publication

0.131

2-methyl-N-(2-[[(2E)-2-[2-[(2-nitrobenzyl)oxy]benzylidene]hydrazinyl]carbonyl]phenyl)benzamide

Escherichia coli

at pH 7.5 and 25°C

0.058

4-(5-[(Z)-[1-(3,4-dimethylphenyl)-3,5-dioxopyrazolidin-4-ylidene]methyl]furan-2-yl)-N-(1,3-thiazol-2-yl)benzenesulfonamide

Escherichia coli

at pH 7.5 and 25°C

0.0348

4-[(3,5-dioxo-1,2,6-thiadiazinan-4-ylidene)methyl]phenyl 2,3-diphenylquinoxaline-6-carboxylate

Escherichia coli

at pH 7.5 and 25°C

8.7

-

3-phosphohydroxypyruvate reduction

9.7

-

alpha-ketoglutarate reduction

additional information

-

7.5

8.5

9.5

-

oxidation of D-3-phosphoglycerate

5.5 - 8.5

-

Tris buffer and phosphate buffer

6 - 8.5

-

activity drops sharply above pH 8.5

25

assay at

25

-

assay at

287525, 287530

45 - 50

-

reduction, but enzyme is thermally unstable, therefore assay is performed at 37°C

-

663605, 667649, 667663, 687564, 711203, 739512, 762672

Uniprot

metabolism

first step of L-serine biosynthesis

metabolism

enzyme in the L-serine biosynthetic pathway

163000

-

sedimentation equilibrium analysis

165000

-

gel filtration

44000

-

4 * 44000, SDS-PAGE, each subunit is divided into 3 separate domains

287545, 287547

tetramer

additional information

9 entries

purified recombinant selenomethionine-labeled enzyme, in presence of 2 mM NAD+ and 5 mM 2-ketoglutarate, euqilibration against precipitation solution containing 1.18-1.25 M ammonium sulfate and 100 mM potassium phosphate, pH 6.4, 18°C, 1-2 weeks, X-ray diffraction structure determination and analysis at 2.24 A resolution

-

-

287525, 287530, 287545, 287546, 287547

A143A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

A144V

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

A374V

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

D317A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

D386A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

E299A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

E302A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

E307A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

E345A

site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme

E360A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

E387A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

G145V

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

G336V

G336V/G337V

changing glycine residues 336 and 337 to valine affect the sensitivity of the enzyme to inhibition by L-serine but not the extent of inhibition. The decrease in sensitivity is caused primarily by a decrease in the affinity of the enzyme for L-serine. The mutations also affect the domain rotation of the subunits in response to L-serine binding

G337V

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

G349V

site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme

G362V

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

H335A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

H344A

site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme

K141A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

K141R

kcat/KM for 2-oxoglutarate is 4.8fold lower than the value for the wild-type enzyme, kcat/KM for 3-phosphooxypyruvate is 2.6fold higher than the value for the wild-type enzyme

K311A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

N190A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, no protein expression

N303A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

N303A/K311A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

N346A

site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme

N364A

site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme

P348A

site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme

Q298A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

Q301A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

Q361A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

Q375A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

R338A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

R347A

site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme

S107A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

S107A/S111A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

S111A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

S111A/K311A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

S296A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

S316A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

S323A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

S373A

site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

T297A

site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme

W139F/E360W

exhibits catalytic activity comparable to that of the native enzyme and is effectively inhibited by L-serine. The only fluorescence signal of the mutant is due to the single tryptophan at position 360. Pre-steady state analysis of binding of inhibitor serine shows that each serine binding interface produces an integrated fluorescent signal

711203

H344A

-

50% inhibition at 0.8 mM L-serine

H344A/N364A

-

no inhibition by L-serine

N346A

-

50% inhibition at 6 mM L-serine

N346A/H344A

-

no inhibition by L-serine

N346A/N364A

-

no inhibition by L-serine

N364A

-

50% inhibition at 48 mM L-serine

W139F/E360W/G294V

-

placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis. Pre-steady state analysis demonstrate that serine binds to two forms of the free enzyme, E and E*. Conversion of Gly-336 to valine has its main effect on the Kd of serine binding to one form of the free enzyme (E) while maintaining the cooperativity of binding observed in the native enzyme

724307

W139F/E360W/G336V

-

placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis. Pre-steady state analysis demonstrate that serine binds to two forms of the free enzyme, E and E*. Conversion of Gly-294 to valine eliminates a rate limiting conformational change that follows serine binding to E. The conformational change between the two forms of free enzyme is maintained, but the Hill coefficient for cooperativity is significantly lowered

724307

additional information

5.5 - 7.5

-

stable

287525

8.5 - 9.5

-

unstable

287525

45

-

3 min, 40% loss of reductase activity

dithiothreitol increases stability

-

-75°C, 15% glycerol, purified stable for months

-

4°C, purified stable for weeks

-

-

Ni-NTA column chromatography and gel filtration

PGDH is expressed in Escherichia coli and purified using 5'-AMP-Sepharose affinity chromatography

recombinant selenomethionine-labeled enzyme from strain B834

-

-

287525, 287530, 287545, 287546, 287548, 287551

diverse mutants expressed in Escherichia coli

-

-

expressed in Escherichia coli BL21(DE3) cells

expression as selenomethionine-labeled enzyme in strain B834

-

-

diverse mutants expressed in Escherichia coli

-

287550, 287551

overexpression of serA gene, that codes for the enzyme, in Escherichia coli strain JM105

-

Sugimoto, E.; Pizer, L.I.

The mechanism of end product inhibition of serine biosynthesis. I. purification and kinetics of phosphoglycerate dehydrogenase

J. Biol. Chem.

243

2081-2089

1968

Escherichia coli

4384871

Pizer, L.I.; Sugimoto, E.

3-Phosphoglycerate dehydrogenase (Escherichia coli)

Methods Enzymol.

17B

325-331

1971

Escherichia coli

-

287534

Winicov, I.

Stereospecificity of hydrogen transfer by phosphoglycerate dehydrogenase

Biochim. Biophys. Acta

397

288-293

1975

Escherichia coli

168922

287538

Tobey, K.L.; Grant, G.A.

The nucleotide sequence of the serA gene of Escherichia coli and the amino acid sequence of the encoded protein, D-3-phosphoglycerate dehydrogenase

J. Biol. Chem.

261

12179-12183

1986

Escherichia coli

3017965

287545

Schuller, D.J.; Grant, G.A.; Banaszak, L.J.

The allosteric ligand site in the Vmax-type cooperative enzyme phosphoglycerate dehydrogenase

Nat. Struct. Biol.

2

69-76

1995

Escherichia coli

7719856

Zhao, G.; Winkler, M.E.

A novel alpha-ketoglutarate reductase activity of the serA-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12, and its possible implications for human 2-hydroxyglutaric aciduria

J. Bacteriol.

178

232-239

1996

Escherichia coli

8550422

287547

Grant, G.A.; Schuller, D.J.; Banaszak, L.J.

A model for the regulation of D-3-phosphoglycerate dehydrogenase, a Vmax-type allosteric enzyme

Protein Sci.

5

34-41

1996

Escherichia coli

8771194

Al-Rabiee, R.; Zhang, Y.; Grant, G.A.

The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase

J. Biol. Chem.

271

23235-23238

1996

Escherichia coli

8798520

287550

Grant, G.A.; Hu, Z.; Xu, X.L.

Amino acid residue mutations uncouple cooperative effects in Escherichia coli D-3-phosphoglycerate dehydrogenase

J. Biol. Chem.

276

17844-17850

2001

Escherichia coli

11278587

287551

Grant, G.A.; Kim, S.J.; Xu, X.L.; Hu, Z.

The contribution of adjacent subunits to the active sites of D-3-phosphoglycerate dehydrogenase

J. Biol. Chem.

274

5357-5361

1999

Escherichia coli

10026144

656083

Grant, G.A.; Hu, Z.; Xu, X.L.

Cofactor binding to Escherichia coli D-3-phosphoglycerate dehydrogenase induces multiple conformations which alter effector binding

J. Biol. Chem.

277

39548-39553

2002

Escherichia coli

12183470

663605

Liberles, J.S.; Thorolfsson, M.; Martinez, A.

Allosteric mechanisms in ACT domain containing enzymes involved in amino acid metabolism

Amino Acids

28

1-12

2005

Escherichia coli (P0A9T0), Escherichia coli

15662561

Grant, G.A.; Hu, Z.; Xu, X.L.

Identification of amino acid residues contributing to the mechanism of cooperativity in Escherichia coli D-3-phosphoglycerate dehydrogenase

Biochemistry

44

16844-16852

2005

Escherichia coli (P0A9T0), Escherichia coli

16363798

667663

Thompson, J.R.; Bell, J.K.; Bratt, J.; Grant, G.A.; Banaszak, L.J.

Vmax regulation through domain and subunit changes. The active form of phosphoglycerate dehydrogenase

Biochemistry

44

5763-5773

2005

Escherichia coli (P0A9T0), Escherichia coli

15823035

Dey, S.; Hu, Z.; Xu, X.L.; Sacchettini, J.C.; Grant, G.A.

The effect of hinge mutations on effector binding and domain rotation in Escherichia coli D-3-phosphoglycerate dehydrogenase

J. Biol. Chem.

282

18418-18426

2007

Escherichia coli (P0A9T0), Escherichia coli

17459882

711203

Burton, R.L.; Chen, S.; Xu, X.L.; Grant, G.A.

Transient kinetic analysis of the interaction of L-serine with Escherichia coli D-3-phosphoglycerate dehydrogenase reveals the mechanism of V-type regulation and the order of effector binding

Biochemistry

48

12242-12251

2009

Escherichia coli (P0A9T0), Escherichia coli

19924905

724307

Grant, G.A.

Transient kinetic analysis of L-serine interaction with Escherichia coli D-3-phosphoglycerate dehydrogenase containing amino acid mutations in the hinge regions

Biochemistry

50

2900-2906

2011

Escherichia coli

21391703

738070

Deng, H.; Chen, C.; Sun, C.; Wei, C.

Construction and characterization of Escherichia coli D-3-phosphoglycerate dehydrogenase mutants with feedback-inhibition relief

Chin. J. Biotechnol.

32

468-477

2016

Escherichia coli

28853268

Wang, Q.; Qi, Y.; Yin, N.; Lai, L.

Discovery of novel allosteric effectors based on the predicted allosteric sites for Escherichia coli D-3-phosphoglycerate dehydrogenase

PLoS ONE

9

e94829

2014

Escherichia coli (P0A9T0), Escherichia coli

24733054

Xu, X.L.; Grant, G.A.

Determinants of substrate specificity in D-3-phosphoglycerate dehydrogenase. Conversion of the M. tuberculosis enzyme from one that does not use alpha-ketoglutarate as a substrate to one that does

Arch. Biochem. Biophys.

671

218-224

2019

Escherichia coli (P0A9T0), Escherichia coli, Mycobacterium tuberculosis (P9WNX3), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv (P9WNX3), Escherichia coli K12 (P0A9T0)

31344342