A C⋅As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters

Abstract

Arsenic is the most widespread environmental toxin. Substantial amounts of pentavalent organoarsenicals have been used as herbicides, such as monosodium methylarsonic acid (MSMA), and as growth enhancers for animal husbandry, such as roxarsone (4-hydroxy-3-nitrophenylarsonic acid) [Rox(V)]. These undergo environmental degradation to more toxic inorganic arsenite [As(III)]. We previously demonstrated a two-step pathway of degradation of MSMA to As(III) by microbial communities involving sequential reduction to methylarsonous acid [MAs(III)] by one bacterial species and demethylation from MAs(III) to As(III) by another. In this study, the gene responsible for MAs(III) demethylation was identified from an environmental MAs(III)-demethylating isolate, Bacillus sp. MD1. This gene, termed arsenic inducible gene (arsI), is in an arsenic resistance (ars) operon and encodes a nonheme iron-dependent dioxygenase with C·As lyase activity. Heterologous expression of ArsI conferred MAs(III)-demethylating activity and MAs(III) resistance to an arsenic-hypersensitive strain of Escherichia coli, demonstrating that MAs(III) demethylation is a detoxification process. Purified ArsI catalyzes Fe 2+ -dependent MAs(III) demethylation. In addition, ArsI cleaves the C·As bond in trivalent roxarsone and other aromatic arsenicals. ArsI homologs are widely distributed in prokaryotes, and we propose that ArsI-catalyzed organoarsenical degradation has a significant impact on the arsenic biogeocycle. To our knowledge, this is the first report of a molecular mechanism for organoarsenic degradation by a C·As lyase.herbicide resistance | growth promoter degradation

Arsenic Methylation Dynamics in a Rice Paddy Soil Anaerobic Enrichment Culture

“…Rapid demethylation of methylated As has been proposed as an explanation for why environmental abundances of methylated arsenic remain low. 43 Arsenic demethylation pathways by aerobes have been characterized 44,45 and a C−As lyase has been identified. 43 Studies of As demethylation under anaerobic conditions have yielded conflicting results, however.…”

Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A

“…Operon composition usually is comprised of at least arsR, arsC (encoding arsenate reductase), and either an arsB or acr3 gene [coding for different proteins involved in As(III) extrusion from the cytoplasm]. Depending on the organism, additional ars operon elements can include arsA, which codes for an ATPase that associates with ArsB, enabling the latter to use ATP to energize the extrusion of As(III) (4); arsD, coding for a protein that can exhibit weak repressor activity, but with a primary function currently viewed as arsenic metallochaperone activity (5-7); arsH, which was recently shown to encode an organoarsenical oxidase capable of oxidizing trivalent methylated and aromatic arsenicals (8); arsI, encoding an alternative As(V) reductase that differs from ArsC (9); another arsI gene, encoding a C-As lyase (10); arsO, encoding a putative flavin-binding monooxygenase (11); arsP, encoding a putative membrane permease (12,13); and arsTX, encoding a thioredoxin system transcribed along with an arsRC2 fusion gene (14). Gene duplication within these operons has been observed (15), as is the case with Agrobacterium tumefaciens 5A (Fig.…”

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

“…MD1 lacking the C-terminus from Glu125 to the end was used in this study and is simply termed arsI because those residues are not essential for enzyme activity. 27 The arsI gene was amplified from Bacillus sp. MD1 genomic DNA 27 with an annealing temperature of 65 °C using Pfu Turbo DNA polymerase (Agilent Technologies, Santa Clara, CA), with forward primer 5′-GGGG CATATG AAATATGCGCATGTGGGTCTT-3′ (NdeI site underlined) and reverse primer 5′-AAAAG GAATTC AA-CAGTTGTCTTTGTGTAG-3′ ( Eco RI site underlined), double-digested by NdeI and Eco RI (New England BioLabs, Ipswich, MA) and cloned into vector plasmid pET28a (Novagen, Merck KGaA, Darmstadt, Germany), generating pET28a- arsI encoding ArsI with an N-terminal 6-histidine tag (Table S1).…”

“…Rox(III) was freshly prepared as described previously. 27 ArsI activity was assayed in a buffer consisting of 0.1 M MOPS, 0.15 M NaCl (pH 7.2) containing 1 mM cysteine, 3 mM TCEP, and 0.1 mM Fe(II). The reaction was initiated by addition of 200 μ M Rox(III), and assayed at 30 °C with shaking at 200 rpm for 3 h. The reaction mixture then was filtered through a 3-kDa cutoff Amicon Ultrafilter to remove protein.…”

“…In addition, ArsI cleaves the C–As bond in a wide range of trivalent aromatic arsenicals, including Rox(III), Nit(III), and p -ASA(III). 27 As of April 2017, 1305 putative ArsI orthologs were identified in 963 aerobic bacterial species by a BLink (Basic Local Alignment Search Tool Link to Protein Alignments and Structures) search, suggesting that arsI genes are widely distributed in the oxic environment. Degradation of environmental organoarsenicals has been also observed under anaerobic condition.…”