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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Biochemical characterization of ArsI: a novel C-As lyase for degradation of environmental organoarsenicals

Pawitwar, Shashank Suryakant 26 June 2017 (has links)
Organoarsenicals such as methylarsenical methylarsenate (MAs(V)) and aromatic arsenicals including roxarsone (4-hydroxy-3-nitrophenylarsenate or Rox(V)) have been extensively used as an herbicide and growth enhancers in animal husbandry, respectively. They undergo environmental degradation to more toxic inorganic arsenite (As(III)) that contaminates crops and drinking water. We previously identified a bacterial gene (arsI) responsible for aerobic MAs(III) demethylation. The gene product, ArsI, is a Fe(II)-dependent extradiol dioxygenase that cleaves the carbon-arsenic (C-As) bond in MAs(III) and trivalent aromatic arsenicals. The objective of this study was to elucidate the ArsI mechanism. Using isothermal titration calorimetry, we determined the dissociation constants (Kd) and ligand-to-protein stoichiometries (N) of ArsI for Fe(II), MAs(III) and aromatic phenyl arsenite. Using a combination of methods including chemical modification, site-directed mutagenesis, and fluorescent spectroscopy, we demonstrated that amino acid residues predicted to participate in Fe(II)-binding (His5-His62-Glu115) and substrate binding (Cys96-Cys97) are all involved in catalysis. Finally, the products of Rox(III) degradation were identified as As(III) and 4-hydroxy-2-nitrophenol, demonstrating that ArsI is a dioxygenase that incorporates one oxygen atom from dioxygen into the carbon and the other to the arsenic to catalyze the cleavage of the C-As bond. These results augment our understanding of the mechanism of this novel C-As lyase.

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