Mutagenesis of a nucleotide-binding site of an anion-translocating ATPase.
The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli. The operon encodes three structural genes arsA, arsB, and arsC. The anion pump requires only two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and was demonstrated to bind ATP by photoaffinity labeling with [alpha-32P]ATP. Analysis of the amino acid sequence deduced from the nucleotide sequence of the arsA gene suggests that the ArsA protein contains two potential nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. A combination of site-directed and bisulfite mutagenesis was used to alter the glycine-rich region of the N-terminal putative nucleotide-binding sequence G15KGGVGKTS23. Four mutant proteins (G18----D, G18----R, G20----S, and T22----I) were analyzed. Strains bearing the mutated plasmids were all arsenite sensitive and were unable to extrude arsenite. Each purified mutant protein lacked oxyanion-stimulated ATPase activity and ATP binding. These results suggest that the N-terminal sequence is part of a nucleotide-binding domain required for catalysis.
Karkaria
C E
CE
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201.
Chen
C M
CM
Rosen
B P
BP
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000838:Anions; D001151:Arsenic / Q000378:metabolism / Q000494:pharmacology; D018053:Arsenites; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007473:Ion Channels / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D009876:Operon; D010777:Photochemistry; D010957:Plasmids; D011815:R Factors / Q000235:genetics; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D000838:Anions; D018053:Arsenites; D007473:Ion Channels; D009711:Nucleotides; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; C015001:arsenite; D001151:Arsenic