1dbv

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spinqualitylabelsall models 1dbv, resolution 2.5Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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+

Overview

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.

About this Structure

1DBV is a Single protein structure of sequence from Geobacillus stearothermophilus with SO4 and NAD as ligands. Active as Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating), with EC number 1.2.1.12 Full crystallographic information is available from OCA.

Reference

A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+., Didierjean C, Rahuel-Clermont S, Vitoux B, Dideberg O, Branlant G, Aubry A, J Mol Biol. 1997 May 16;268(4):739-59. PMID:9175858

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