Arsenic is classified as a carcinogen by the U.S, Environmental Protection Agency. More recently arsenic trioxide (Trisenox) has been approved for use as a chemotherapeutic agent for the treatment of acute promyelocytic leukemia. The goal of this research is the elucidation of the molecular mechanisms in members of two families of arsenate reductases. The arsenate (As(V)) reductase found in many bacteria is typified by the ArsC enzyme encoded by the (ars) operon found on the clinically-isolated E. coli resistance plasmid R773. Eukaryotes have pathways for arsenic detoxification that arose through convergent evolution. The ACR2 gene of Saccharomyces cerevisiae encodes an arsenate reductase required for arsenate resistance in yeast but is not an ArsC homologue. Specific aims include: 1) Structure/function relationships in the arsenate reductases: Residues in the E. coli ArsC and S. cerevisiae Acr2p involved in function will be identified such as anion binding sites and residues involved in catalysis. 2) Interaction of ArsC with glutaredoxin: Glutaredoxin 2 (Grx2) is the most efficient hydrogen donor for ArsC. The contact points between Grx2 and ArsC will be determined by a combination of biochemical and genetic approaches including crystallography, NMR and florescence spectroscopy. Similar methods will be applied to the interaction of Acr2p and glutaredoxin. 3) Characterization of the Leishmania major LmAcr2p arsenate reductase: As one consequence of the war on terrorism American soldiers are more and more frequently returning from Iraq with "Baghdad boil" -better known as leishmaniasis. The first-drug for treatment of this re-emerging infectious disease is the pentalvalent antimonial drug Pentostam, and Pentostam resistance is becoming a major clinical problem. We propose that LmAcr2p plays a role biotransformation of Pentostam to its active form by reducing Sb(V) to Sb(III). 4) The structure two eukaryotic arsenate reductases will be determined: The structure two eukaryotic arsenate reductases, ScAcr2p from S. cerevisiae and LmAcr2p from L. major will be determined using x-ray crystallography. These proteins can be produced in large amounts and are easily purified for crystallization trials. LmAcr2p and the G83A mutant of ScAcr2p are both small monomeric proteins that are good candidates for structural studies by NMR spectroscopy.

R01GM052216

1995-04-01

2008-11-30