Arsenite Methyltransferase Diversity and Optimization of Methylation Efficiency

Arsenic is methylated by arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferases (ArsMs). ArsM crystal structures show three domains (an N-terminal SAM binding domain (A domain), a central arsenic binding domain (B domain), and a C-terminal domain of unknown function (C domain)). In this study, we performed a comparative analysis of ArsMs and found a broad diversity in structural domains. The differences in the ArsM structure enable ArsMs to have a range of methylation efficiencies and substrate selectivities. Many small ArsMs with 240–300 amino acid residues have only A and B domains, represented by RpArsM from Rhodopseudomonas palustris. These small ArsMs have higher methylation activity than larger ArsMs with 320–400 residues such as Chlamydomonas reinhardtii CrArsM, which has A, B, and C domains. To examine the role of the C domain, the last 102 residues in CrArsM were deleted. This CrArsM truncation exhibited higher As(III) methylation activity than the wild-type enzyme, suggesting that the C-terminal domain has a role in modulating the rate of catalysis. In addition, the relationship of arsenite efflux systems and methylation was examined. Lower rates of efflux led to higher rates of methylation. Thus, the rate of methylation can be modulated in multiple ways

Organoarsenical Biotransformations by <i>Shewanella putrefaciens</i>

Microbes play a critical role in the global arsenic biogeocycle. Most studies have focused on redox cycling of inorganic arsenic in bacteria and archaea. The parallel cycles of organoarsenical biotransformations are less well characterized. Here we describe organoarsenical biotransformations in the environmental microbe <i>Shewanella putrefaciens</i>. Under aerobic growth conditions, <i>S. putrefaciens</i> reduced the herbicide MSMA (methylarsenate or MAs­(V)) to methylarsenite (MAs­(III)). Even though it does not contain an <i>arsI</i> gene, which encodes the ArsI C–As lyase, <i>S. putrefaciens</i> demethylated MAs­(III) to As­(III). It cleaved the C–As bond in aromatic arsenicals such as the trivalent forms of the antimicrobial agents roxarsone (Rox­(III)), nitarsone (Nit­(III)) and phenylarsenite (PhAs­(III)), which have been used as growth promoters for poultry and swine. <i>S. putrefaciens</i> thiolated methylated arsenicals, converting MAs­(V) into the more toxic metabolite monomethyl monothioarsenate (MMMTAs­(V)), and transformed dimethylarsenate (DMAs­(V)) into dimethylmonothioarsenate (DMMTAs­(V)). It also reduced the nitro groups of Nit­(V), forming <i>p</i>-aminophenyl arsenate (<i>p</i>-arsanilic acid or <i>p</i>-AsA­(V)), and Rox­(III), forming 3-amino-4-hydroxybenzylarsonate (3A4HBzAs­(V)). Elucidation of organoarsenical biotransformations by <i>S. putrefaciens</i> provides a holistic appreciation of how these environmental pollutants are degraded

Identification of Catalytic Residues in the As(III) <i>S</i>-Adenosylmethionine Methyltransferase

The enzyme As­(III) <i>S</i>-adenosylmethionine methyltransferase (EC 2.1.1.137) (ArsM or AS3MT) is found in members of every kingdom, from bacteria to humans. In these enzymes, there are three conserved cysteine residues at positions 72, 174, and 224 in the CmArsM orthologue from the thermophilic eukaryotic alga <i>Cyanidioschyzon</i> sp. 5508. Substitution of any of the three led to loss of As­(III) methylation. In contrast, a C72A mutant still methylated trivalent methylarsenite [MAs­(III)]. Protein fluorescence of a single-tryptophan mutant reported binding of As­(III) or MAs­(III). As­(GS)₃ and MAs­(GS)₂ bound significantly faster than As­(III), suggesting that the glutathionylated arsenicals are preferred substrates for the enzyme. Protein fluorescence also reported binding of Sb­(III), and the purified enzyme methylated and volatilized Sb­(III). The results suggest that all three cysteine residues are necessary for the first step in the reaction, As­(III) methylation, but that only Cys174 and Cys224 are required for the second step, methylation of MAs­(III) to dimethylarsenite [DMAs­(III)]. The rate-limiting step was identified as the conversion of DMAs­(III) to trimethylarsine, and DMAs­(III) accumulates as the principal product

The Structure of an As(III) <i>S</i>‑Adenosylmethionine Methyltransferase with 3‑Coordinately Bound As(III) Depicts the First Step in Catalysis

Arsenic is a ubiquitous environmental toxic substance and a Class 1 human carcinogen. Arsenic methylation by the enzyme As­(III) <i>S</i>-adenosylmethionine (SAM) methyltransferase (ArsM in microbes or AS3MT in animals) detoxifies As­(III) in microbes but transforms it into more toxic and potentially more carcinogenic methylated species in humans. We previously proposed a reaction pathway for ArsM/AS3MT that involves initial 3-coordinate binding of As­(III). To date, reported structures have had only 2-coordinately bound trivalent arsenicals. Here we report a crystal structure of CmArsM from <i>Cyanidioschyzon</i> sp.5508 in which As­(III) is 3-coordinately bound to three conserved cysteine residues with a molecule of the product <i>S</i>-adenosyl-l-homocysteine bound in the SAM binding site. We propose that this structure represents the first step in the catalytic cycle. In a previously reported SAM-bound structure, a disulfide bond is formed between two conserved cysteine residues. Comparison of these two structures indicates that there is a conformational change in the N-terminal domain of CmArsM that moves a loop to allow formation of the 3-coordinate As­(III) binding site. We propose that this conformational change is an initial step in the As­(III) SAM methyltransferase catalytic cycle

Arsenic Binding and Transfer by the ArsD As(III) Metallochaperone

ArsD is a metallochaperone that delivers trivalent metalloids [As(III) or Sb(III)] to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Interaction with ArsD increases the affinity of ArsA for As(III), conferring resistance to environmental concentrations of arsenic. Previous genetic analysis suggested that ArsD residues Cys12, Cys13, and Cys18 are involved in the transfer of As(III) to ArsA. Here X-ray absorption spectroscopy was used to show that As(III) is coordinated with three sulfur atoms, consistent with the three cysteine residues forming the As(III) binding site. Two single-tryptophan derivatives of ArsD exhibited quenching of intrinsic protein fluorescence upon binding of As(III) or Sb(III), which allowed estimation of the rates of binding and affinities for metalloids. Substitution of Cys12, Cys13, or Cys18 decreased the affinity for As(III) more than 10-fold. Reduced glutathione greatly increased the rate of binding of As(III) to ArsD but did not affect binding of As(III) to ArsA. This suggests that in vivo cytosolic As(III) might be initially bound to GSH and transferred to ArsD and then to ArsAB, which pumps the metalloid out of the cell. The As(III) chelator dimercaptosuccinic acid did not block the transfer from ArsD to ArsA, consistent with channeling of the metalloid from one protein to the other, as opposed to release and rebinding of the metalloid. Finally, transfer of As(III) from ArsD to ArsA occurred in the presence of MgATP at 23 °C but not at 4 °C. Neither MgADP nor MgATP-γ-S could replace MgATP. These results suggest that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle

Experimental and Theoretical Characterization of Arsenite in Water: Insights into the Coordination Environment of As−O

Long-term exposure to arsenic in drinking water has been linked to cancer of the bladder, lungs, skin, kidney, nasal passages, liver, and prostate in humans. It is therefore important to understand the structural aspects of arsenic in water, as hydrated arsenic is most likely the initial form of the metalloid absorbed by cells. We present a detailed experimental and theoretical characterization of the coordination environment of hydrated arsenite. XANES analysis confirms As(III) is a stable redox form of the metalloid in solution. EXAFS analysis indicate, at neutral pH, arsenite has a nearest-neighbor coordination geometry of approximately 3 As−O bonds at an average bond length of 1.77 Å, while at basic pH the nearest-neighbor coordination geometry shifts to a single short As−O bond at 1.69 Å and two longer As−O bonds at 1.82 Å. Long-range ligand scattering is present in all EXAFS samples; however, these data could not be fit with any degree of certainty. There is no XAS detectable interaction between As and antimony, suggesting they are not imported into cells as a multinuclear complex. XAS results were compared to a structural database of arsenite compounds to confirm that a 3 coordinate As−O complex for hydrated arsenite is the predominate species in solution. Finally, quantum chemical studies indicate arsenite in solution is solvated by 3 water molecules. These results indicate As(OH)3 as the most stable structure existing in solution at neutral pH; thus, ionic As transport does not appear to be involved in the cellular uptake process

The 1.4 Å Crystal Structure of the ArsD Arsenic Metallochaperone Provides Insights into Its Interaction with the ArsA ATPase

Arsenic is a carcinogen that tops the Superfund list of hazardous chemicals. Bacterial resistance to arsenic is facilitated by ArsD, which delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump. Here we report the structure of the arsenic metallochaperone ArsD at 1.4 Å and a model for its binding of metalloid. There are two ArsD molecules in the asymmetric unit. The overall structure of the ArsD monomer has a thioredoxin fold, with a core of four β-strands flanked by four α-helices. Based on data from structural homologues, ArsD was modeled with and without bound As(III). ArsD binds one arsenic per monomer coordinated with the three sulfur atoms of Cys12, Cys13, and Cys18. Using this structural model, an algorithm was used to dock ArsD and ArsA. The resulting docking model provides testable predictions of the contact points of the two proteins and forms the basis for future experiments

Pathway of Human AS3MT Arsenic Methylation

A synthetic gene encoding human As­(III) <i>S</i>-adenosylmethionine (SAM) methyltransferase (hAS3MT) was expressed, and the purified enzyme was characterized. The synthetic enzyme is considerably more active than a cDNA-expressed enzyme using endogenous reductants thioredoxin (Trx), thioredoxin reductase (TR), NADPH, and reduced glutathione (GSH). Each of the seven cysteines (the four conserved residues, Cys32, Cys61, Cys156, and Cys206, and nonconserved, Cys72, Cys85, and Cys250) was individually changed to serine. The nonconserved cysteine derivates were still active. None of the individual C32S, C61S, C156S, and C206S derivates were able to methylate As­(III). However, the C32S and C61S enzymes retained the ability to methylate MAs­(III). These observations suggest that Cys156 and Cys206 play a different role in catalysis than that of Cys32 and Cys61. A homology model built on the structure of a thermophilic orthologue indicates that Cys156 and Cys206 form the As­(III) binding site, whereas Cys32 and Cys61 form a disulfide bond. Two observations shed light on the pathway of methylation. First, binding assays using the fluorescence of a single-tryptophan derivative indicate that As­(GS)₃ binds to the enzyme much faster than inorganic As­(III). Second, the major product of the first round of methylation is MAs­(III), not MAs­(V), and remains enzyme-bound until it is methylated a second time. We propose a new pathway for hAS3MT catalysis that reconciles the hypothesis of Challenger ((1947) <i>Sci. Prog.</i>, <i>35</i>, 396–416) with the pathway proposed by Hayakawa et al. ((2005) <i>Arch. Toxicol</i>., <i>79</i>, 183–191). The products are the more toxic and more carcinogenic trivalent methylarsenicals, but arsenic undergoes oxidation and reduction as enzyme-bound intermediates

Selective Methylation by an ArsM <i>S</i>‑Adenosylmethionine Methyltransferase from Burkholderia gladioli GSRB05 Enhances Antibiotic Production

Arsenic methylation contributes to the formation and diversity of environmental organoarsenicals, an important process in the arsenic biogeochemical cycle. The arsM gene encoding an arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferase is widely distributed in members of every kingdom. A number of ArsM enzymes have been shown to have different patterns of methylation. When incubated with inorganic As(III), Burkholderia gladioli GSRB05 has been shown to synthesize the organoarsenical antibiotic arsinothricin (AST) but does not produce either methylarsenate (MAs(V)) or dimethylarsenate (DMAs(V)). Here, we show that cells of B. gladioli GSRB05 synthesize DMAs(V) when cultured with either MAs(III) or MAs(V). Heterologous expression of the BgarsM gene in Escherichia coli conferred resistance to MAs(III) but not As(III). The cells methylate MAs(III) and the AST precursor, reduced trivalent hydroxyarsinothricin (R-AST-OH) but do not methylate inorganic As(III). Similar results were obtained with purified BgArsM. Compared with ArsM orthologs, BgArsM has an additional 37 amino acid residues in a linker region between domains. Deletion of the additional 37 residues restored As(III) methylation activity. Cells of E. coli co-expressing the BgarsL gene encoding the noncanonical radical SAM enzyme that catalyzes the synthesis of R-AST-OH together with the BgarsM gene produce much more of the antibiotic AST compared with E. coli cells co-expressing BgarsL together with the CrarsM gene from Chlamydomonas reinhardtii, which lacks the sequence for additional 37 residues. We propose that the presence of the insertion reduces the fitness of B. gladioli because it cannot detoxify inorganic arsenic but concomitantly confers an evolutionary advantage by increasing the ability to produce AST