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Characterization of the metalloactivation domain of an arsenite/antimonite resistance pump
Author(s) -
Ruan Xiang,
Bhattacharjee Hiranmoy,
Rosen Barry P.
Publication year - 2008
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2007.06049.x
Subject(s) - metalloid , arsenite , mutant , biology , binding site , efflux , biochemistry , mutagenesis , chemistry , arsenic , metal , gene , organic chemistry
Summary 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.