title: The catalytic site of chloroplastic NADP-dependent malate dehydrogenase contains a His/Asp pair.
abstract: Plant chloroplastic NADP-malate dehydrogenase is unique among malate dehydrogenases because of its reductive activation in the light and cofactor specificity. In this paper, the role of His229 in sorghum leaf protein has been investigated by site-directed mutagenesis. His229 was replaced by Asn and Gln, both mutations yielding an inactive protein. The role of a conserved Asp (Asp201) as a possible partner of His229 in catalysis has been studied by the same approach. Both Asp mutants (D201A, D201N) were only slightly active and were essentially characterized by a dramatically increased Km for oxaloacetate (45-80-fold). pH dependence of catalytic rates revealed differences between the two Asp mutants. These results demonstrate that, in sorghum leaf NADP-dependent malate dehydrogenase, His229 is involved in catalysis in interaction with Asp201.
authors.affiliation: Institut de Biotechnologie des Plantes, Université Paris-Sud, France.
authors.forename: M
authors.initials: M
authors.lastname: Lemaire
authors.forename: M
authors.initials: M
authors.lastname: Miginiac-Maslow
authors.forename: P
authors.initials: P
authors.lastname: Decottignies
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010944:Plants / Q000201:enzymology*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006639:Histidine; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)