title: Involvement of conserved glycine residues, 229 and 234, of Vibrio harveyi aldehyde dehydrogenase in activity and nucleotide binding.
abstract: The involvement of two conserved glycine residues (Gly229 and Gly234) in activity and nucleotide binding in Vibrio harveyi aldehyde dehydrogenase (ALDH) have been investigated. Each of the glycine residues has been mutated to alanine and the mutant ALDHs have been expressed in Escherichia coli and specifically labelled with [35S]methionine. The G229A mutant was inactive with either NADP+ or NAD+ as coenzyme and did not bind to 2',5'-ADP Sepharose, indicating a complete loss of nucleotide affinity. In contrast, the G234A mutant showed a high affinity for 2',5'-ADP Sepharose. Purified G234A mutant showed similar kinetic properties to the native enzyme including a pre-steady-state burst of NADPH; however, the Michaelis constants for NAD+ and NADP+ were increased by 3- to 9-fold, showing that the mutation had an effect on saturation of the enzyme with NAD(P)+. These data are consistent with the structure for the nucleotide binding domain of Vh.ALDH being similar to that of class 3 or class 2 mammalian ALDHs which differ from the classical nucleotide binding domain found in most dehydrogenases.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, H3G 1Y6, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Vedadi
authors.forename: A
authors.initials: A
authors.lastname: Vrielink
authors.forename: E
authors.initials: E
authors.lastname: Meighen
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D016415:Sequence Alignment; D014733:Vibrio / Q000201:enzymology*
chemical_list: D009711:Nucleotides; D000444:Aldehyde Dehydrogenase