title: Probing the coenzyme specificity of glyceraldehyde-3-phosphate dehydrogenases by site-directed mutagenesis.
abstract: By combining our knowledge of the crystal structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the sequence of the photosynthetic NADP-dependent GAPDH of the chloroplast, two particular amino acid residues were predicted as the principal determinants of differing coenzyme specificity. By use of site-directed mutagenesis, the amino acids Leu 187 and Pro 188 of GAPDH from Bacillus stearothermophilus have been replaced with Ala 187 and Ser 188, which occur in the sequence from the chloroplast enzyme. The resulting mutant was shown to be catalytically active not only with its natural coenzyme NAD but also with NADP, thus confirming the initial hypothesis. This approach has not only enabled us to alter the coenzyme specificity by minimal amino acid changes but also revealed factors that control the relative affinity of the enzyme for NAD and NADP.
authors.affiliation: Laboratoire d'Enzymologie et de Génie Génétique, URA CNRS, Faculté des Sciences, Vandoeuvre les Nancy, France.
authors.forename: C
authors.initials: C
authors.lastname: Corbier
authors.forename: S
authors.initials: S
authors.lastname: Clermont
authors.forename: P
authors.initials: P
authors.lastname: Billard
authors.forename: T
authors.initials: T
authors.lastname: Skarzynski
authors.forename: C
authors.initials: C
authors.lastname: Branlant
authors.forename: A
authors.initials: A
authors.lastname: Wonacott
authors.forename: G
authors.initials: G
authors.lastname: Branlant
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002736:Chloroplasts / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism
chemical_list: D003067:Coenzymes; D004269:DNA, Bacterial; D009243:NAD; D009249:NADP; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases