Arsenicals are one of the oldest treatments for a variety of human disorders. Although infamous for its toxicity, arsenic is paradoxically a therapeutic agent that has been used since ancient times for the treatment of multiple diseases. In the 1970s, arsenic trioxide (ATO), the active ingredient in a traditional Chinese medicine, was shown to produce dramatic remission of acute promyelocytic leukemia (APL) similar to the effect of all-trans retinoic acid (ATRA). The discovery of the pentavalent arsenic-containing antibiotic arsinothricin (AST), which is effective against multidrug-resistant pathogens, illustrates the future potential of this new class of organoarsenical antibiotics. The goal of this dissertation is to identify the AST biosynthetic pathway and the mechanism of transport of AST and related compounds into and out of the cells of bacteria. In the first study, three genes, arsQML, in an arsenic resistance operon were shown to be a biosynthetic gene cluster responsible for synthesis of AST and its precursor, hydroxyarsinothricin (2-amino-4-(dihydroxyarsinoyl) butanoate or AST-OH). The viii arsL gene product is a noncanonical radical S-adenosylmethionine (SAM) enzyme that is predicted to transfer the 3-amino-3-carboxypropyl (ACP) group from SAM to the arsenic atom in inorganic arsenite, forming the reduced trivalent form of AST-OH (R-AST-OH), which is methylated by the arsM gene product, an arsenic SAM methyltransferase, to produce the reduced trivalent form of AST (R-AST). ArsQ is an efflux permease that was proposed to transport AST or related species out of the cells. In a follow-up study, B. gladioli arsQ was expressed in Escherichia coli and shown to confer resistance to AST and related arsenicals. Cells of E. coli expressing arsQ transport R-AST-OH and R-AST, with little transport of their pentavalent forms. Transport is independent of cellular energy and appears to be equilibrative. A structural homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R-AST-OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy-independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.
