Asp45 is a Mg2+ ligand in the ArsA ATPase.
The ATPase activity of ArsA, the catalytic subunit of the plasmid-encoded, ATP-dependent extrusion pump for arsenicals and antimonials in Escherichia coli, is allosterically activated by arsenite or antimonite. Magnesium is essential for ATPase activity. To examine the role of Asp45, mutants were constructed in which Asp45 was changed to Glu, Asn, or Ala. Cells expressing these mutated arsA genes lost arsenite resistance to varying degrees. Purified D45A and D45N enzymes were inactive. The purified D45E enzyme exhibited approximately 5% of the wild type activity with about a 5-fold decrease in affinity for Mg2+. Intrinsic tryptophan fluorescence was used to probe Mg2+ binding. ArsA containing only Trp159 exhibited fluorescence enhancement upon the addition of MgATP, which was absent in D45N and D45A. As another measure of conformation, limited trypsin digestion was used to estimate the surface accessibility of residues in ArsA. ATP and Sb(III) synergistically protected wild type ArsA from trypsin digestion. Subsequent addition of Mg2+ increased trypsin sensitivity. D45N and D45A remained protected by ATP and Sb(III) but lost the Mg2+ effect. D45E exhibited an intermediate Mg2+ response. These results indicate that Asp45 is a Mg2+-responsive residue, consistent with its function as a Mg2+ ligand.
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000965:Antimony / Q000378:metabolism; D053501:Arsenite Transporting ATPases; D018053:Arsenites / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D016384:Consensus Sequence; D004357:Drug Synergism; D004926:Escherichia coli; D016623:Ion Pumps; D008024:Ligands; D008274:Magnesium / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence