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Biochemical Characterization of ArsI: A Novel C-As Lyase for Degradation of Environmental Organoarsenicals.

ABSTRACT: Organoarsenicals such as the methylarsenical methylarsenate (MAs(V)) and aromatic arsenicals including roxarsone (4-hydroxy-3-nitrobenzenearsenate 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 demethylation of methylarsenite (MAs(III)). The gene product, ArsI, is an Fe(II)-dependent extradiol dioxygenase that cleaves the carbon-arsenic (C-As) bond in MAs(III) and in trivalent aromatic arsenicals. The objective of this study was to elucidate the ArsI mechanism. Using isothermal titration calorimetry, we determined the dissociation constants and ligand-to-protein stoichiometry of ArsI for Fe(II), MAs(III), and aromatic phenylarsenite. 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 involved in catalysis. Finally, the products of Rox(III) degradation were identified as As(III) and 2-nitrohydroquinone, demonstrating that ArsI is a dioxygenase that incorporates one oxygen atom from dioxygen into the carbon and the other to the arsenic to catalyze cleavage of the C-As bond. These results augment our understanding of the mechanism of this novel C-As lyase.

SUBMITTER: Pawitwar SS

PROVIDER: S-EPMC5870903 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Biochemical Characterization of ArsI: A Novel C-As Lyase for Degradation of Environmental Organoarsenicals.

Pawitwar Shashank S SS Nadar Venkadesh S VS Kandegedara Ashoka A Stemmler Timothy L TL Rosen Barry P BP Yoshinaga Masafumi M

Environmental science & technology 20170922 19

Organoarsenicals such as the methylarsenical methylarsenate (MAs(V)) and aromatic arsenicals including roxarsone (4-hydroxy-3-nitrobenzenearsenate 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 demethylation of methylarsenite (MAs(III)). ...[more]

PMID: 28936873

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Project description:We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.

Dataset Information

Biochemical Characterization of ArsI: A Novel C-As Lyase for Degradation of Environmental Organoarsenicals.

Publications

Biochemical Characterization of ArsI: A Novel C-As Lyase for Degradation of Environmental Organoarsenicals.

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