Mutation of Conserved Residues in the NADP(H)-Binding Domain of the Proton Translocating Pyridine Nucleotide Transhydrogenase ofEscherichia coli

Possible NADP(H)-binding sites of the β subunit of the pyridine nucleotide transhydrogenase ofEscherichia coliwere examined by site-directed mutagenesis. The sequence of the β subunit at positions 314–350 showed several features typical of NADP(H)-binding sites. Mutation of βGly314, the first glycine residue of the GXGXXV motif, and of βArg350, 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 βArg350 mutants was not due to loss of ability to bind NADP(H). Several residues (βVal319, βGly337, βHis345, and βArg350) were 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 βαβ nucleotide-binding folds, in the sequence of the β 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 β subunit, a possible nucleotide-binding site, prevented assembly and incorporation of the truncated enzyme into the cytoplasmic membrane ofE. coli.