INVOLVEMENT OF CONSERVED GLYCINE RESIDUES, 229 AND 234, OF VIBRIO-HARVEYI ALDEHYDE DEHYDROGENASE IN ACTIVITY AND NUCLEOTIDE-BINDING

The involvement of two conserved glycine residues (Gly229 and Gly234) in activity and nucleotide binding in Vibrio harveyi aldehyde dehydrog enase (ALDH) have been investigated. Each of the glycine residues has been mutated to alanine and the mutant ALDHs have been expressed in Es cherichia coli and specifically labelled with [S-35]methionine. The G2 29A mutant was inactive with either NADP(+) or NAD(+) as coenzyme and did not bind to 2',5'-ADP Sepharose, indicating a complete loss of nuc leotide 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 presteady-state burst of NADPH; however, the Michaelis constants for NAD(+) and NADP(+) were in creased by 3- to 9-fold, showing that the mutation had an effect on sa turation of the enzyme with NAD(P)(+). These data are consistent with the structure for the nucleotide binding domain of Vh.ALDH being simil ar to that of class 3 or class 2 mammalian ALDHs which differ from the classical nucleotide binding domain found in most dehydrogenases. (C) 1997 Academic Press.