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 nuc leotide transhydrogenase of Escherichia coli were examined by site-dir ected mutagenesis. The sequence of the beta subunit at positions 314-3 50 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 sub strate 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 His3 45, 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 incorporati on into the membrane. The GXGXXG motif, typical of beta alpha beta nuc leotide-binding folds, in the sequence of the beta subunit at position s 274-279 was mutated without causing major changes in transhydrogenas e 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 incorporat ion of the truncated enzyme into the cytoplasmic membrane of E. coli. (C) 1997 Academic Press.