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 betaGly314, the first glycine residue of the GXGXXV motif, and of betaArg350, 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 betaArg350 mutants was not due to loss of ability to bind NADP(H). Several residues (betaVal319, betaGly337, betaHis345, and betaArg350) 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 beta alphabeta 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.
Bragg
P D
PD
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Glavas
N A
NA
Hou
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014357:Trypsin
D009249:NADP; D009250:NADP Transhydrogenases; D014357:Trypsin