PDBsum entry 1nq5

Oxidoreductase

PDB id: 1nq5

Contents

Protein chains

334 a.a. *

Ligands

SO4 ×4

NAD ×4

Waters ×604

* Residue conservation analysis

PDB id:

1nq5

Name:

Oxidoreductase

Title:

Glyceraldehyde-3-phosphate dehydrogenase mutant with cys 149 replaced by ser complexed with NAD+

Structure:

Glyceraldehyde 3-phosphate dehydrogenase. Chain: o, q, a, c. Synonym: gapdh. Engineered: yes. Mutation: yes

Source:

Geobacillus stearothermophilus. Organism_taxid: 1422. Gene: gap. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PDB file)

Resolution:

2.11Å R-factor: 0.198 R-free: 0.249

Authors:

C.Didierjean,C.Corbier,M.Fatih,F.Favier,S.Boschi-Muller,G.Branlant, A.Aubry

Key ref:

C.Didierjean et al. (2003). Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus with NAD and D-glyceraldehyde 3-phosphate. J Biol Chem, 278, 12968-12976. PubMed id: 12569100 DOI: 10.1074/jbc.M211040200

Date:

21-Jan-03 Release date: 22-Apr-03

Protein chains

P00362  (G3P_GEOSE) -  Glyceraldehyde-3-phosphate dehydrogenase from Geobacillus stearothermophilus

Seq: 335 a.a.

Struc: 334 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.2.1.12 - glyceraldehyde-3-phosphate dehydrogenase (phosphorylating).
• Pathway: Glyceraldehyde-3-phosphate Dehydrogenase (phosphorylating)
• Reaction: D-glyceraldehyde 3-phosphate + phosphate + NAD⁺ = (2R)-3-phospho- glyceroyl phosphate + NADH + H⁺

D-glyceraldehyde 3-phosphate + phosphate (Bound ligand (Het Group name = NAD) corresponds exactly) + NAD(+) = (2R)-3-phospho- glyceroyl phosphate + NADH + H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M211040200

J Biol Chem 278:12968-12976 (2003)

PubMed id: 12569100

Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus with NAD and D-glyceraldehyde 3-phosphate.

C.Didierjean, C.Corbier, M.Fatih, F.Favier, S.Boschi-Muller, G.Branlant, A.Aubry.

ABSTRACT

The crystal structure of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus was solved in complex with its cofactor, NAD, and its physiological substrate, D-glyceraldehyde 3-phosphate (D-G3P). To isolate a stable ternary complex, the nucleophilic residue of the active site, Cys(149), was substituted with alanine or serine. The C149A and C149S GAPDH ternary complexes were obtained by soaking the crystals of the corresponding binary complexes (enzyme.NAD) in a solution containing G3P. The structures of the two binary and the two ternary complexes are presented. The D-G3P adopts the same conformation in the two ternary complexes. It is bound in a non-covalent way, in the free aldehyde form, its C-3 phosphate group being positioned in the P(s) site and not in the P(i) site. Its C-1 carbonyl oxygen points toward the essential His(176), which supports the role proposed for this residue along the two steps of the catalytic pathway. Arguments are provided that the structures reported here are representative of a productive enzyme.NAD.D-G3P complex in the ground state (Michaelis complex).

Selected figure(s)

Figure 1.
Fig. 1. Schematic representation of the postulated catalytic mechanism of phosphorylating GAPDHs. The catalytic mechanism can be divided into two steps. In the acylation step, Cys149 and His176 form an ion pair in holo-GAPDH (a). This decreases the pK[app] of Cys149, thus facilitating the thiolate attack toward the C-1 of D-G3P. The role of His 176 is also to stabilize the binding of the substrate in the Michaelis complex GAPDH·NAD·D-G3P (b), the thiohemiacetal intermediate (c), and the thioacylenzyme intermediate (d). His176 also plays a role as a base catalyst facilitating the hydride transfer from the thiohemiacetal toward the nicotinamidium of NAD (c). In the phosphorylating step, the binding of inorganic phosphate to the thioacylenzyme is followed by its nucleophilic attack toward the thioacyl intermediate (d), which leads via a sp3-phosphorylated intermediate (e) to the formation and release of 1,3-dPG (f). His176 is postulated to stabilize the tetrahedral intermediate (e) and to facilitate, as an acid (d) or base (e) catalyst, the 1,3-dPG formation. The exchange cofactor step, which consists of NADH release prior to NAD and inorganic phosphate binding, remains controversial (32-40). R' represents the adenine-ribose-phosphate-phosphate-ribose part of the cofactor, NAD. R represents the CH(OH)COPO[3] part of the substrate, D-G3P.

Figure 5.
Fig. 5. Stereoview of the active site in monomer O of the ternary complexes. A, ternary complex I (C149A mutant GAPDH); B, ternary complex II (C149A mutant GAPDH). The residues that interact with the D-G3P molecule are labeled.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 12968-12976) copyright 2003.

Figures were selected by an automated process.