Mutation of conserved residues in the NADP(H)-binding domain of the proton translocating pyridine nucleotide transhydrogenase of Escherichia coli

Possible NADP(H)-binding sites of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli were examined by site-directed mutagenesis. The sequence of the beta subunit at positions 314-350 showed several features typical of NADP(H)-binding sites, Mutation of beta Gly314, the first glycine residue of the GXGXXV motif, and of beta Arg350, which probably interacts with the 2'-phosphate of the substrate NADP(H), resulted in drastic loss of enzyme activity. The loss of activity in the beta Arg350 mutants was not due to loss of ability to bind NADP(H). Several residues (beta Val319, beta Gly337, beta His345, and beta Arg350) mere mutated to make the sequence more similar to that of a NAD(H)-binding site. The introduction of multiple mutations resulted in improper assembly of the enzyme and decreased incorporation into the membrane. The GXGXXG motif, typical of beta alpha beta nucleotide-binding folds, in the sequence of the beta subunit at positions 274-279 was mutated without causing major changes in transhydrogenase activities, It is unlikely to be part of a nucleotide-binding domain. Deletion of the carboxy-terminal 32 amino acids of the beta subunit, a possible nucleotide-binding site, prevented assembly and incorporation of the truncated enzyme into the cytoplasmic membrane of E. coli. (C) 1997 Academic Press.