AC·As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters Masafumi Yoshinaga1 and Barry P. Rosen Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199 Edited by Jerome Nriagu, University of Michigan, Ann Arbor, MI, and accepted by the Editorial Board April 16, 2014 (received for review February 18, 2014) Arsenic is the most widespread environmental toxin. Substantial More complex pentavalent aromatic arsenicals such as roxarsone amounts of pentavalent organoarsenicals have been used as herbi- [4-hydroxy-3-nitrophenylarsonic acid, Rox(V)] have been largely cides, such as monosodium methylarsonic acid (MSMA), and as used since the middle of the 1940s as antimicrobial growth pro- growth enhancers for animal husbandry, such as roxarsone moters for poultry and swine to control Coccidioides infections (4-hydroxy-3-nitrophenylarsonic acid) [Rox(V)]. These undergo envi- and improve weight gain, feed efficiency, and meat pigmentation ronmental degradation to more toxic inorganic arsenite [As(III)]. We (8, 9). These aromatic arsenicals are largely excreted unchanged previously demonstrated a two-step pathway of degradation of and introduced into the environment when chicken litter is ap- MSMA to As(III) by microbial communities involving sequential reduc- plied to farmland as fertilizer (8). Pentavalent organoarsenicals tion to methylarsonous acid [MAs(III)] by one bacterial species and are relatively benign and less toxic than inorganic arsenicals; demethylation from MAs(III) to As(III) by another. In this study, however, aromatic (8–10) and methyl (11, 12) arsenicals are de- the gene responsible for MAs(III) demethylation was identified from graded into more toxic inorganic forms in the environment, which Bacillus an environmental MAs(III)-demethylating isolate, sp. MD1. may contaminate the foods and water supplies. Although micro- arsI This gene, termed arsenic inducible gene ( ), is in an arsenic resis- bial degradation of environmental organoarsenicals has been ars tance ( ) operon and encodes a nonheme iron-dependent dioxyge- documented (8, 9, 11, 13), no molecular details of the reaction · nase with C As lyase activity. Heterologous expression of ArsI have been reported. We recently demonstrated that a microbial conferred MAs(III)-demethylating activity and MAs(III) resistance Escherichia coli community in Florida golf course soil carries out a two-step to an arsenic-hypersensitive strain of , demonstrat- pathway of MSMA reduction and demethylation (14). Here we ing that MAs(III) demethylation is a detoxification process. Purified + report the isolation of an environmental methylarsonous acid ArsI catalyzes Fe2 -dependent MAs(III) demethylation. In addition, [MAs(III)]-demethylating bacterium Bacillus sp. MD1 (for ArsI cleaves the C·As bond in trivalent roxarsone and other aromatic “MAs(III) demethylating”) from Florida golf course soil and the arsenicals. ArsI homologs are widely distributed in prokaryotes, and cloning of the gene, termed arsenic inducible gene (arsI), re- we propose that ArsI-catalyzed organoarsenical degradation has a significant impact on the arsenic biogeocycle. To our knowl- sponsible for MAs(III) demethylation. The gene product, ArsI, is nonheme iron-dependent dioxygenase with C·As lyase activity. ArsI edge, this is the first report of a molecular mechanism for organo- · arsenic degradation by a C·As lyase. cleaves the C As bond in a wide range of trivalent organoarsenicals, including the trivalent roxarsone [Rox(III)], into As(III), which herbicide resistance | growth promoter degradation Significance he metalloid arsenic is the most common environmental toxic Tsubstance, entering the biosphere primarily from geochemical Organoarsenicals are used as herbicides, pesticides, antimicro- sources, but also through anthropogenic activities (1). Arsenic is bial growth promoters, and chemical warfare agents. Envi- a group 1 human carcinogen that ranks first on the Agency for ronmental organoarsenicals are microbially degraded, but the Toxic Substances and Disease Registry Priority List of Hazardous molecular mechanisms of breakdown are unknown. We pre- Substances (www.atsdr.cdc.gov/SPL/index.html). Microbial arsenic viously identified a two-step pathway of degradation in- volving sequential reduction and C·As bond cleavage. Here we transformations create a global arsenic biogeocycle (1). These bio- report cloning of the gene and characterization of the gene transformations include redox cycles between the relatively innoc- product for a C·As lyase, ArsI, a member of the family of type I uous pentavalent arsenate and the considerably more toxic and SCIENCES extradiol dioxygenases. ArsI is the only enzyme shown to be carcinogenic trivalent arsenite (2, 3). In addition, many microbes, ENVIRONMENTAL arsM involved in degradation of the reduced forms of the herbicide both prokaryotic and eukaryotic, have genes for inorganic monosodium methylarsonic acid and the antimicrobial growth arsenite [As(III)] S-adenosylmethionine methyltransferases that promoter roxarsone. As arsI genes are widely distributed in methylate inorganic As(III) to mono-, di-, and tri-methylated bacteria, ArsI-catalyzed organoarsenic degradation is proposed species (4, 5). The genes encoding arsenic transforming enzymes to have an impact on the arsenic biogeocycle. are widely distributed, and these arsenic biotransformations have been proposed to play significant roles in the arsenic biogeocycle Author contributions: M.Y. and B.P.R. designed research; M.Y. performed research; M.Y. and in remodeling the terrain in volcanic areas such as Yellowstone contributed new reagents/analytic tools; M.Y. and B.P.R. analyzed data; and M.Y. and B.P.R. National Park and regions of the world with high amounts of arsenic wrote the paper. in soil and water such as West Bengal and Bangladesh (3, 6). The authors declare no conflict of interest. Arsenicals, both inorganic and organic, have been used in This article is a PNAS Direct Submission. J.N. is a guest editor invited by the Editorial agriculture in the United States for more than a century (7). Board. Data deposition: The sequences reported in this paper have been deposited in the Gen- Historically, the use of inorganic arsenical pesticides/herbicides Bank database [accession nos. KF899846 (Bacillus sp. MD1 16S ribosomal RNA gene, par- has been largely replaced by methylated arsenicals such as tial sequence) and KF899847 (Bacillus sp. MD1, partial genome sequence)]. monosodium methylarsonic acid (MSMA), which is still in use as 1To whom correspondence should be addressed. E-mail: [email protected]. an herbicide for turf maintenance on golf courses, sod farms, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. highway rights of way, and for weed control on cotton fields (7). 1073/pnas.1403057111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1403057111 PNAS | May 27, 2014 | vol. 111 | no. 21 | 7701–7706 Downloaded by guest on September 27, 2021 strongly suggests that the environmental pentavalent phenyl- 2,3-dioxygenase encoded by akbC from Rhodococcus sp. strain arsenicals such as Rox(V) also undergo a two-step pathway of DK17 (20) and 2,3-dihydroxybiphenyl 1,2-dioxygenase encoded by sequential reduction and ArsI-catalyzed dearylation, in analogy bphC from Pseudomonas sp. strain KKS102 (21) (Fig. S5). Al- with the demethylation of MSMA by a microbial community. though the N- and C-terminal domains are structurally similar to Thus, ArsI-catalyzed C·As bond cleavage is a newly identified each other, only the C-terminal domain binds metal and functions mechanism for degradation of organoarsenical herbicides and in catalysis (19). The divalent metal binding site of these dioxy- antimicrobial growth promoters. genase contains a triad of three charged amino acid residues. A Results homology search using Protein Basic Local Alignment Search Bacillus Tool identified His5-His62-Glu115 as a putative metal binding site Isolation of a MAs(III)-Demethylating from Golf Course Soil. in the Bacillus ArsI, which corresponds to those of AkbC and We previously reported the isolation of methylarsonic acid Burkholderia BphC (Fig. S5). His5 is replaced by a glutamine residue in the [MAs(V)]-reducing bacterium sp. MR1 and the Thermomonospora curvata Streptomyces putative ArsI orthologs from DSM MAs(III)-demethylating bacterium sp. MD1 from Streptomyces coelicolor Florida golf course soil (14). Together, these two activities result in 43183 and A3 (2) (Fig. S5). degradation of the herbicide MSMA. In this study, a second A Basic Local Alignment Search Tool Link to Protein Align- bacterial strain capable of MAs(III) demethylation was isolated. ments and Structures search identified nearly 650 putative ArsI Like Streptomyces sp. MD1, this isolate demonstrated no MAs orthologs in 487 bacterial species, with no representatives in other Bacillus (V) transformation when cultured alone (Fig. S1A,curve2),but kingdoms. In the ArsI sequence there are four vicinal nearly completely transformed MAs(V) into As(III) when cocul- cysteine pairs. One of the four (Cys96-Cys97 in the Bacillus ArsI) tured with Burkholderia sp. MR1 (Fig. S1A, curve 1), suggesting that is conserved in all putative ArsI orthologs, and the other three the isolate possesses MAs(III) demethylating activity. In confirma- pairs in Bacillus ArsI, located near the C terminus, are not conserved tion,