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A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+.
J Mol Biol. 1997 May 16; 268(4):739-59.JM

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.

Authors+Show Affiliations

Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, IFR Protéines, Université Henri Poincaré Nancy I (URA CNRS 809), Vandoeuvre-lès-Nancy, France.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9175858

Citation

Didierjean, C, et al. "A Crystallographic Comparison Between Mutated Glyceraldehyde-3-phosphate Dehydrogenases From Bacillus Stearothermophilus Complexed With Either NAD+ or NADP+." Journal of Molecular Biology, vol. 268, no. 4, 1997, pp. 739-59.
Didierjean C, Rahuel-Clermont S, Vitoux B, et al. A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+. J Mol Biol. 1997;268(4):739-59.
Didierjean, C., Rahuel-Clermont, S., Vitoux, B., Dideberg, O., Branlant, G., & Aubry, A. (1997). A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+. Journal of Molecular Biology, 268(4), 739-59.
Didierjean C, et al. A Crystallographic Comparison Between Mutated Glyceraldehyde-3-phosphate Dehydrogenases From Bacillus Stearothermophilus Complexed With Either NAD+ or NADP+. J Mol Biol. 1997 May 16;268(4):739-59. PubMed PMID: 9175858.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+. AU - Didierjean,C, AU - Rahuel-Clermont,S, AU - Vitoux,B, AU - Dideberg,O, AU - Branlant,G, AU - Aubry,A, PY - 1997/5/16/pubmed PY - 1997/5/16/medline PY - 1997/5/16/entrez SP - 739 EP - 59 JF - Journal of molecular biology JO - J. Mol. Biol. VL - 268 IS - 4 N2 - 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. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/9175858/A_crystallographic_comparison_between_mutated_glyceraldehyde_3_phosphate_dehydrogenases_from_Bacillus_stearothermophilus_complexed_with_either_NAD+_or_NADP+_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(97)90998-0 DB - PRIME DP - Unbound Medicine ER -