A CRYSTALLOGRAPHIC COMPARISON BETWEEN MUTATED GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASES FROM BACILLUS-STEAROTHERMOPHILUS COMPLEXED WITH EITHER NAD(+) OR NADP(+)

Mutations have been introduced in the cytosolic glyceraldehyde-3-phosp hate dehydrogenase (GAPDH) from Bacillus stearothermophilus in order t o 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 alignmen t 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 con siderati ons (D32G). Both mutants exhibit a dual-cofactor selectivity at the ad vantage of either NAD (B-S) or NADP (D32G-S). In order to analyse the cofactor-binding site plasticity at the molecular level, crystal struc tures of these mutants have been solved, when complexed with either NA D(+) (D32G-Sn, resolution 2.5 Angstrom, R=13.9%; B-Sn, 2.45 Angstrom, 19.3%) or NADP(+) (D32G-Sp, 2.2 Angstrom, 19.2%; B-Sp, 2.5 Angstrom, 1 4.4%). The four refined models are very similar to that of the wild-ty pe GAPDH and as expected resemble more closely the hole form than the apo form. In the B-S mutant, the wild-type low affinity for NADP(+) se ems to be essentially retained because of repulsive electrostatic cont acts between the extra 2'-phosphate and the unchanged carboxylate grou p of residue D32. Such an antideterminant effect is not well compensat ed by putative attractive interactions which had been expected to aris e from the newly-introduced side-chains. In this mutant, recognition o f NAD(+) is slightly affected with respect to that known on the wild-t ype, 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 l ong-range and/or second-layer effects, not easily predictable from vis ual inspection of three-dimensional structures, need to be taken into account for designing a true ''chloroplastic-like'' mutant of cytosoli c 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 wildtype. The strong alteration of the affinity for NAD(+) obvi ously proceeds from the suppression of the two wild-type hydrogen bond s between the adenosine 2'- and 5'-hydroxyl positions and the D32 carb oxylate group. As expected, the efficient recognition of NADP(+) is pa rtly promoted by the removal of intra-subunit electrostatic repulsion (D32G) and inter-subunit steric hindrance (L187A, P188S). Another inte resting feature of the reshaped NADP(+)-binding site is provided by th e 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, thi s result clearly demonstrates that an absolute need for a salt-bridge involving the 2'-phosphate is not required to switch the cofactor sele ctivity from NAD to NADP. In fact, as it is the case in this mutant, o nly a moderately polar hydrogen bond can be sufficient to make the ext ra 2'-phosphate of NADP(+) well recognized by a protein environment. ( C) 1997 Academic Press Limited.