Characterization of the metalloactivation domain of an arsenite/antimonite resistance pump.
The ArsAB extrusion pump encoded by the ars operon of Escherichia coli plasmid R773 confers resistance to the toxic trivalent metalloids arsenite [As(III)] and antimonite [Sb(III)]. The ArsA ATPase, the catalytic subunit of the pump, has two homologous halves, A1 and A2. At the interface of these two halves are two nucleotide-binding domains and a metalloid-binding domain. Cys-113 and Cys-422 have been shown to form a high-affinity metalloid binding site. The crystal structure of ArsA shows two other bound metalloid atoms, one liganded to Cys-172 and His-453, and the other liganded to His-148 and Ser-420. The contribution of those putative metalloid sites was examined. There was little effect of mutagenesis of residues His-148 and Ser-420 on metalloid binding. However, a C172A ArsA mutant and C172A/H453A double mutant exhibited significantly decreased affinity for Sb(III). These results suggest first that there is only a single high-affinity metalloid binding site in ArsA, and second that Cys-172 controls the affinity of this site for metalloid and hence the efficiency of metalloactivation of the ArsAB efflux pump.
Ruan
Xiang
X
Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, MI 48201, USA.
Bhattacharjee
Hiranmoy
H
Rosen
Barry P
BP
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000965:Antimony / Q000378:metabolism* / Q000633:toxicity; D018053:Arsenites / Q000378:metabolism* / Q000633:toxicity; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D016623:Ion Pumps / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007477:Ions / Q000378:metabolism*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
D018053:Arsenites; D029968:Escherichia coli Proteins; D016623:Ion Pumps; D007477:Ions; D009097:Multienzyme Complexes; C061374:antimonite; D000255:Adenosine Triphosphate; D000965:Antimony; C486481:ArsAB ATPase, E Coli; C015001:arsenite