My Ph.D. thesis focuses on mechanisms of arsenic detoxification in bacteria, is centered on three major objectives, mainly, the metalloid-stimulated ArsA ATPase, and arsenite-specific transporter, Acr3.;Objective 1: Mutagenesis study of deviant Walker A motif of ArsA ATPase. ArsA is composed of homologous N-terminal (A1) and C-terminal (A2) halves that are connected by a short linker. Each contains a nucleotide binding domain (NBD), and both NBDs are required for ATPase activity and metalloid transport. The metalloid binding domain located at the A1-A2 interface diametrically opposite to the NBDs modulates ATPase activity. Two stretches of residues, D142TAPTGH148T in A1 and D447TAPTGH 453T in A2, physically connect the metalloid binding domain to the A1 and A2 NBDs, respectively. The metalloid binding domain is composed of three metalloid atoms, each of which is coordinated by two protein ligands, one of which is donated by A1 and the other by A2. Thus the metalloid atoms serve as the "molecular glue" that brings the two halves of the protein together, activating catalysis. However, it is not clear that how the NBDs interact with each other during activated catalysis. To answer this question, I focused on the conserved lysine residues (Lys16 and Lys335) in both NBDs, which have been suggested to play a role in subunit interaction in the homologue NifH.;Alteration of Lys16 did not show any significant sensitivity to metalloids, but Lys335 mutants were considerably less resistant to arsenite than wild type. Azido-ATP labeling studies indicated that while both K16Q and K335Q bind nucleotide at both NBD1 and NBD2, the binding affinity decreases in the order: wild-type>K16Q>K335Q. More interestingly, while K16Q hydrolyzes ATP at either NBD1 or NBD2, K335Q ArsA does not exhibit ATP hydrolysis at either site. Based on the above data, we propose that akin to NifH, Lys335 is involved in charge stabilization of the bound nucleotide at NBD1, and is consequently involved in the activation of ATPase activity. Lys16 is probably slightly distant to have a strong electrostatic interaction with the negative charge of the bound nucleotide at NBD2, and consequently, alteration of Lys16 does not have a drastic effect on ATPase activity. A similar phenomenon was observed with the conserved aspartates, Asp142 and Asp447, located in A1 and A2 signal transduction domain, respectively. While alteration of Asp142 resulted in loss of Mg2+ binding to NBD1, Asp447 was found not to be nearly as critical for Mg2+ binding as Asp142.;Objective 2: Characterization of ArsA1/2 from the novel ars operon in Alkaliphilus metalliredigens QYMF. Much less is known about the second family of arsenite carriers, Acr3. Members of this family are found in bacteria, archaea and fungi. The identification of the novel ars operon in A. metalliredigens QYMF which contains arsA1, arsA2, and Amacr3 is thought provoking. This is the first time that, instead of arsB, acr3 is found to coexist with arsA in an operon. I have cloned this novel operon and showed that it confers arsenite resistance upon expression in an arsenic-sensitive strain of E. coli. I also determined that the purified A. metalliredigens ArsA1-ArsA2 complex shares similar characteristics with its R773 homologue.;Objective 3: Membrane topology of the Acr3 arsenite transporter from Alkaliphilus metalliredigens QYMF. To date however, no mechanistic data is available on any member of the Acr3 family. In order to be able to rationally study the function of a transport protein, it is important to have a basic understanding of the structure of the transporter. Here I use scanning cysteine accessibility mutagenesis (SCAM) to study the membrane topology of AmAcr3. Thirty-four single cysteine were introduced by mutagenesis in "cysteineless" (C27/91S) backbone, and the sideness of these cysteine were probed with membrane permeable and membrane impermeable thiol-specific reagents, 3-(N-maleimidylpropionyl) biocytin (Biotin maleimide or BM) and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). The data suggested that both the N- and the C-termini are located in the cytoplasm, and a 10-TM model was proposed. More over, energistics study using everted membrane vesicles, it showed that both DeltapH and Deltapsiare required for As (III) transport.
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