Literature summary for 1.2.1.12 extracted from

Schormann, N.; Ayres, C.A.; Fry, A.; Green, T.J.; Banerjee, S.; Ulett, G.C.; Chattopadhyay, D.
Crystal structures of group B Streptococcus glyceraldehyde-3-phosphate dehydrogenase apo-form, binary and ternary complexes (2016), PLoS ONE, 11, e0165917 .

Application

Application	Comment	Organism
drug development	as an essential enzyme for the survival of GBS, GAPDH may be a potential target for developing antibacterial drugs	Streptococcus agalactiae

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of wild-type and mutant enzymes carrying a 20-residue N-terminal tag containing hexa-histidine and a thrombin cleavage sequence in Escherichia coli strain Rosetta (DE3) pLysS	Streptococcus agalactiae

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified wild-type enzyme in apo-form, in a mixed apo/holo-state (2 subunits with bound NAD and two without), and in a ternary complex, and purified mutant C152S enzyme in ternary complex, hanging drop vapor diffusion method, mixing of 15 mg/ml protein in 25 mM HEPES, pH 7.35, 00 mM NaCl, and 5 mM 2-mercaptoethanol with reservoir solution containing 20-28% PEG 4000, 0.1 M MES, pH 6.5, at 22°C, X-ray diffraction structure determination and analysis at 2.0 A resolution	Streptococcus agalactiae

Crystallization (Comment)

Organism

purified wild-type enzyme in apo-form, in a mixed apo/holo-state (2 subunits with bound NAD and two without), and in a ternary complex, and purified mutant C152S enzyme in ternary complex, hanging drop vapor diffusion method, mixing of 15 mg/ml protein in 25 mM HEPES, pH 7.35, 00 mM NaCl, and 5 mM 2-mercaptoethanol with reservoir solution containing 20-28% PEG 4000, 0.1 M MES, pH 6.5, at 22°C, X-ray diffraction structure determination and analysis at 2.0 A resolution

Streptococcus agalactiae

Protein Variants

Protein Variants	Comment	Organism
C152S	site-directed mutagenesis, mutation of the catalytic residue inactivates the enzyme	Streptococcus agalactiae

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	the mammalian enzyme shows negative cooperativity	Streptococcus agalactiae

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
160000	-	recombinant tagged enzyme, gel filtration	Streptococcus agalactiae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
D-glyceraldehyde 3-phosphate + phosphate + NAD+	Streptococcus agalactiae	-	3-phospho-D-glyceroyl phosphate + NADH + H+	-	r

Organism

Organism	UniProt	Comment	Textmining
Streptococcus agalactiae	Q9ALW2	Group B Streptococcus	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta(DE3)pLysS by nickel affinity chromatography and gel filtration	Streptococcus agalactiae

Reaction

Reaction	Comment	Organism	Reaction ID
D-glyceraldehyde 3-phosphate + phosphate + NAD+ = 3-phospho-D-glyceroyl phosphate + NADH + H+	GAPDH-catalyzed phosphorylation of D-G3H takes place in two steps. In the first exergonic reaction the aldehyde group of D-glyceraldehyde 3-phosphate is converted into a carboxylic acid with concomitant reduction of NAD+ to NADH. The energy released by this reaction drives the endergonic second reaction in which a molecule of inorganic phosphate is transferred to the intermediate acid to form the product 3-phospho-D-glyceroyl phosphate. The reaction mechanism involves formation of a covalent bond between the thiol group of a conserved cysteine residue of GAPDH and the carbonyl C-atom of D-glyceraldehyde 3-phosphate resulting in the formation of the hemithioacetal intermediate. A hydride ion is transferred from D-glyceraldehyde 3-phosphate to the cofactor NAD+ to form NADH while oxidation of D-glyceraldehyde 3-phosphate by a water molecule generates a thioester intermediate. In the second step, the thioester is phosphorylated in a nucleophilic attack by an inorganic phosphate ion resulting in the formation of the product 3-phospho-D-glyceroyl phosphate and the release of the thiol-group of the active site cysteine	Streptococcus agalactiae	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
D-glyceraldehyde 3-phosphate + phosphate + NAD+	-	Streptococcus agalactiae	3-phospho-D-glyceroyl phosphate + NADH + H+	-	r

Subunits

Subunits	Comment	Organism
homotetramer	a dimer of dimers, 4 * 44000, about, SDS-PAGE	Streptococcus agalactiae
More	subunits of the dimers form the major interface P, overview. The second largest interface, the R interface, includes residues in the N-terminal domain that interact with NAD+ and loop residues 181-206 in the C-terminal domain of subunit pairs A, C and B, D. The smallest interface, the Q interface, shows limited interactions between residues in the ranges 43-53 and 274-291 of adjacent subunits A, D and B, C	Streptococcus agalactiae

Synonyms

Synonyms	Comment	Organism
GAPDH	-	Streptococcus agalactiae
GBS GAPDH	-	Streptococcus agalactiae
glyceraldehyde-3-phosphate dehydrogenase	-	Streptococcus agalactiae

Cofactor

Cofactor	Comment	Organism	Structure
NAD+	1 NAD+ molecule per subunit, all four subunits have NAD+ bound in the active site, enzyme binding structure of wild-type and mutant C152S enzymes, overview	Streptococcus agalactiae
NADH	-	Streptococcus agalactiae

General Information

General Information	Comment	Organism
additional information	the phosphate group of the substrate is bound to the phosphate site in all four subunits, adenosyl binding pocket structure, comparison of group B Streptococcus ternary complex of enzyme with substrate and cofactor with human structure, comparative structure-function analysis of GBS GAPDH and hGAPDH, conformational changes upon ligand binding, overview. The active site residue Cys152 is positioned between the nicotinamide moiety of NAD+ and the side chain of active site residue His179	Streptococcus agalactiae
physiological function	enzyme GAPDH plays a key role in glycolysis and gluconeogenesis by catalyzing the reversible oxidative phosphorylation of D-glyceraldehyde 3-phosphate to the energy-rich intermediate glyceraldehyde 1,3-bisphosphate	Streptococcus agalactiae