PDBsum entry 3cmc

Oxidoreductase

PDB id: 3cmc

Contents

Protein chains

334 a.a. *

Ligands

SO4 ×18

G3H ×4

NAD ×4

GOL ×8

EDO

Waters ×1740

* Residue conservation analysis

PDB id:

3cmc

Name:

Oxidoreductase

Title:

Thioacylenzyme intermediate of bacillus stearothermophilus phosphorylating gapdh

Structure:

Glyceraldehyde-3-phosphate dehydrogenase. Chain: o, p, q, r. Synonym: gapdh. Engineered: yes

Source:

Bacillus stearothermophilus. Gene: gap. Expressed in: escherichia coli.

Resolution:

1.77Å R-factor: 0.163 R-free: 0.198

Authors:

S.Moniot,C.Vonrhein,G.Bricogne,C.Didierjean,C.Corbier

Key ref:

S.Moniot et al. (2008). Trapping of the Thioacylglyceraldehyde-3-phosphate Dehydrogenase Intermediate from Bacillus stearothermophilus: DIRECT EVIDENCE FOR A FLIP-FLOP MECHANISM. J Biol Chem, 283, 21693-21702. PubMed id: 18480053 DOI: 10.1074/jbc.M802286200

Date:

21-Mar-08 Release date: 17-Jun-08

Protein chains

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

Seq: 335 a.a.

Struc: 334 a.a.

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 (Bound ligand (Het Group name = G3H) corresponds exactly) + phosphate + NAD(+) (Bound ligand (Het Group name = NAD) corresponds exactly) = (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.M802286200

J Biol Chem 283:21693-21702 (2008)

PubMed id: 18480053

Trapping of the Thioacylglyceraldehyde-3-phosphate Dehydrogenase Intermediate from Bacillus stearothermophilus: DIRECT EVIDENCE FOR A FLIP-FLOP MECHANISM.

S.Moniot, S.Bruno, C.Vonrhein, C.Didierjean, S.Boschi-Muller, M.Vas, G.Bricogne, G.Branlant, A.Mozzarelli, C.Corbier.

ABSTRACT

The crystal structure of the thioacylenzyme intermediate of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been solved at 1.8A resolution. Formation of the intermediate was obtained by diffusion of the natural substrate within the crystal of the holoenzyme in the absence of inorganic phosphate. To define the soaking conditions suitable for the isolation and accumulation of the intermediate, a microspectrophotometric characterization of the reaction of GAPDH in single crystals was carried out, following NADH formation at 340 nm. When compared with the structure of the Michaelis complex ( Didierjean, C., Corbier, C., Fatih, M., Favier, F., Boschi-Muller, S., Branlant, G., and Aubry, A. (2003) J. Biol. Chem. 278, 12968-12976 ) the 206-210 loop is shifted and now forms part of the so-called "new P(i)" site. The locations of both the O1 atom and the C3-phosphate group of the substrate are also changed. Altogether, the results provide evidence for the flipping of the C3-phosphate group occurring concomitantly or after the redox step.

Selected figure(s)

Figure 4.
FIGURE 4. Alternate conformations of the 206-212 loop and re-location of the P[i] anion binding site. A, stereoscopic view of the O subunit region spanning residues 205-218. The 206-212 loop is represented in two alternate conformations: in dark gray, the classic conformation delineates the P[i] anion binding site where a sulfate is bound (conformation A). In light gray, the alternate conformation exhibits a shift toward the C3P of the intermediate (conformation B). This motion allows Thr-208 and Gly-209 to participate to the formation of the new P[i] site and thus to the binding of the C3P group of the intermediate. B, superimposition of the 206-212 loop with representative GAPDH structures. The superimposition of the 206-212 loop under its classic conformation (dark gray) with the holo-structure from B. stearothermophilus (in white, pdb code 1gd1 (10)) is reported on the left, and the superimposition of the 206-212 loop under its alternate conformation with the structure of T. maritima GAPDH (in white, pdb code 1hdg (11)) is reported on the right. All structures are represented in schematic mode with their corresponding bound anion in stick mode. To facilitate comparison, both conformations of our model were represented in each superimposition, shading the one that is not considered.

Figure 5.
FIGURE 5. Structural scenario proposed for the catalytic mechanism. Only hydrogen atoms directly involved in the catalytic mechanism are represented. G3P initially binds to the active site of GAPDH with its C3P group located in the P[s] site. Reaction begins through the nucleophilic attack of the Cys-149 thiolate function on the aldehydic carbon C1 of the substrate (step 1), which leads to a tetrahedral intermediate called hemithioacetal (HTA). This step is followed by hydride transfer (step 2), assisted by His-176, from the C1 atom of G3P to the C4 atom of NAD^+. At this stage, C3P is thought to be located in the P[s] site, and the O1 atom of G3P is expected to interact with His-176 (N ) (see "Discussion"). To allow NADH release, the substrate must undergo a conformational change during which the orientation of the O1 atom changes while the C3P group flips toward the new P[i] site (step 3). It results in the loss of the interaction between the substrate and the cofactor required for NADH release (TAE: thioacylenzyme intermediate corresponding to the structure depicted here). NAD^+ enters the active site and likely promotes the relocation of O1 and C3P to their initial position. The P[i] site (either in the classic or new position, indicated as "Pi?") is free to bind an inorganic phosphate for the nucleophilic attack on the thioacylenzyme. This last step is also assisted by His-176 and leads to the formation of 1,3-bisphosphoglycerate. Subsequent release of the product leaves the enzyme in its holo-state, ready to accommodate a new G3P molecule.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 21693-21702) copyright 2008.

Figures were selected by an automated process.