PDBsum entry 1dbv

Oxidoreductase

PDB id: 1dbv

Contents

Protein chains

334 a.a. *

Ligands

SO4 ×8

NAD ×4

Waters ×384

* Residue conservation analysis

PDB id:

1dbv

Name:

Oxidoreductase

Title:

Glyceraldehyde-3-phosphate dehydrogenase mutant with asp 32 replaced by gly, leu 187 replaced by ala, and pro 188 replaced by ser complexed with NAD+

Structure:

Glyceraldehyde-3-phosphate dehydrogenase. Chain: o, p, q, r. Synonym: gapdh. Engineered: yes. Mutation: yes. Other_details: complexed with NAD+

Source:

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

Biol. unit:

Tetramer (from PQS)

Resolution:

2.50Å R-factor: 0.140 R-free: 0.214

Authors:

C.Didierjean,S.Rahuel-Clermont,B.Vitoux,O.Dideberg,G.Branlant,A.Aubry

Key ref:

C.Didierjean et al. (1997). A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+. J Mol Biol, 268, 739-759. PubMed id: 9175858 DOI: 10.1006/jmbi.1997.0998

Date:

20-Dec-96 Release date: 07-Jul-97

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 3 residue positions (black crosses)

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: 10.1006/jmbi.1997.0998

J Mol Biol 268:739-759 (1997)

PubMed id: 9175858

A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+.

C.Didierjean, S.Rahuel-Clermont, B.Vitoux, O.Dideberg, G.Branlant, A.Aubry.

ABSTRACT

Mutations have been introduced in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus in order to convert its cofactor selectivity from a specificity towards NAD into a preference for NADP. In the B-S mutant, five mutations (L33T, T34G, D35G, L187A, P188S) were selected on the basis of a sequence alignment with NADP-dependent chloroplastic GAPDHs. In the D32G-S mutant, two of the five mutations mentioned above (L187A, P188S) have been used in combination with another one designed from electrostatic considerations (D32G). Both mutants exhibit a dual-cofactor selectivity at the advantage of either NAD (B-S) or NADP (D32G-S). In order to analyse the cofactor-binding site plasticity at the molecular level, crystal structures of these mutants have been solved, when complexed with either NAD+ (D32G-Sn, resolution 2.5 A, R = 13.9%; B-Sn, 2.45 A, 19.3%) or NADP+ (D32G-Sp, 2.2 A, 19.2%; B-Sp, 2.5 A, 14.4%). The four refined models are very similar to that of the wild-type GAPDH and as expected resemble more closely the holo form than the apo form. In the B-S mutant, the wild-type low affinity for NADP+ seems to be essentially retained because of repulsive electrostatic contacts between the extra 2'-phosphate and the unchanged carboxylate group of residue D32. Such an antideterminant effect is not well compensated by putative attractive interactions which had been expected to arise from the newly-introduced side-chains. In this mutant, recognition of NAD+ is slightly affected with respect to that known on the wild-type, because mutations only weakly destabilize hydrogen bonds and van der Waals contacts originally present in the natural enzyme. Thus, the B-S mutant does not mimic efficiently the chloroplastic GAPDHs, and long-range and/or second-layer effects, not easily predictable from visual inspection of three-dimensional structures, need to be taken into account for designing a true "chloroplastic-like" mutant of cytosolic GAPDH. In the case of the D32G-S mutant, the dissociation constants for NAD+ and NADP+ are practically reversed with respect to those of the wild-type. The strong alteration of the affinity for NAD+ obviously proceeds from the suppression of the two wild-type hydrogen bonds between the adenosine 2'- and 3'-hydroxyl positions and the D32 carboxylate group. As expected, the efficient recognition of NADP+ is partly promoted by the removal of intra-subunit electrostatic repulsion (D32G) and inter-subunit steric hindrance (L187A, P188S). Another interesting feature of the reshaped NADP+-binding site is provided by the local stabilization of the extra 2'-phosphate which forms a hydrogen bond with the side-chain hydroxyl group of the newly-introduced S188. When compared to the presently known natural NADP-binding clefts, this result clearly demonstrates that an absolute need for a salt-bridge involving the 2'-phosphate is not required to switch the cofactor selectivity from NAD to NADP. In fact, as it is the case in this mutant, only a moderately polar hydrogen bond can be sufficient to make the extra 2'-phosphate of NADP+ well recognized by a protein environment.

Selected figure(s)

Figure 1.
Figure 1. Localization of secondary structures in the schematic representation of the GAPDH subunit from B. stearothermophilus.

Figure 5.
Figure 5. Hydrogen-bond networks in the adenosine-binding subsite of NADP+/GAPDH complexes: monomer O of Sp mutant (a; G. B., S. R.-C. & A. J. Wonacott, unpublished results), D32G-Sp mutant (b) and B-Sp mutant (c).

The above figures are reprinted by permission from Elsevier: J Mol Biol (1997, 268, 739-759) copyright 1997.

Figures were selected by an automated process.