RESUMEN
Arsenic is a naturally existing toxin and carcinogen. As(III) S-adenosylmethionine methyltransferases (AS3MT in mammals and ArsM in microbes) methylate As(III) three times in consecutive steps and play a central role in arsenic metabolism from bacteria to humans. Current assays for arsenic methylation are slow, laborious, and expensive. Here we report the development of two in vitro assays for AS3MT activity that are rapid, sensitive, convenient, and relatively inexpensive and can be adapted for high-throughput assays. The first assay measures As(III) binding by the quenching of the protein fluorescence of a single-tryptophan derivative of an AS3MT ortholog. The second assay utilizes time-resolved fluorescence resonance energy transfer to directly measure the conversion of the AS3MT substrate, S-adenosylmethionine, to S-adenosylhomocysteine catalyzed by AS3MT. These two assays are complementary, one measuring substrate binding and the other catalysis, making them useful tools for functional studies and future development of drugs to prevent arsenic-related diseases.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Metiltransferasas/metabolismo , S-Adenosilmetionina/metabolismo , Cromatografía Líquida de Alta Presión , Activación Enzimática , Escherichia coli/enzimología , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Espectrometría de Masas , Metiltransferasas/análisisRESUMEN
A synthetic gene encoding human As(III) S-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) Sci. Prog., 35, 396-416) with the pathway proposed by Hayakawa et al. ((2005) Arch. Toxicol., 79, 183-191). The products are the more toxic and more carcinogenic trivalent methylarsenicals, but arsenic undergoes oxidation and reduction as enzyme-bound intermediates.
Asunto(s)
Arsenitos/metabolismo , Metiltransferasas/metabolismo , Arsenitos/toxicidad , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Humanos , Metilación , Metiltransferasas/genética , Modelos Moleculares , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
The ArsA ATPase is the catalytic subunit of the ArsAB As(III) efflux pump. It receives trivalent As(III) from the intracellular metallochaperone ArsD. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. A number of arsA genes with multiple mutations were isolated. These were analyzed in more detail by separation into single arsA mutants. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant in yeast two-hybrid assays. Each of the three single ArsA mutants also interacted with wild type ArsD. Only the Q56R ArsA derivative exhibited significant metalloid-stimulated ATPase activity in vitro. Purified Q56R ArsA was stimulated by wild type ArsD and to a lesser degree by the quadruple ArsD derivative. The F120I and D137V ArsAs did not show metalloid-stimulated ATPase activity. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. We predict that mutations in ArsA propagate changes in hydrogen bonding and salt bridges to the ArsA-ArsD interface that affect their interactions.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Escherichia coli/genética , Bombas Iónicas/genética , Chaperonas Moleculares/genética , Complejos Multienzimáticos/genética , Mutación Missense , Mutación Puntual , Adenosina Trifosfatasas/metabolismo , Sustitución de Aminoácidos , Arsénico/metabolismo , Catálisis , Proteínas de Escherichia coli/metabolismo , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Bombas Iónicas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Simulación del Acoplamiento Molecular , Complejos Multienzimáticos/metabolismo , Mutagénesis , Plásmidos , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.
Asunto(s)
Arsénico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Arseniato Reductasas/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/análisis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , ADN de Hongos/genética , ADN de Hongos/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Unión Proteica , Conformación Proteica/efectos de los fármacos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
Arsenic is one the most toxic environmental substances. Arsenic is ubiquitous in water, soil and food, and ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. Arsenic(III) S-adenosylmethionine methyltransferases (AS3MT in animals and ArsM in microbes) are key enzymes of arsenic biotransformation, catalyzing the methylation of inorganic arsenite to give methyl, dimethyl and trimethyl products. Arsenic methyltransferases are found in members of every kingdom from bacteria to humans (EC 2.1.1.137). In the human liver, hAS3MT converts inorganic arsenic into more toxic and carcinogenic forms. CrArsM, an ortholog of hAS3MT from the eukaryotic green alga Chlamydomonas reinhardtii, was purified by chemically synthesizing the gene and expressing it in Escherichia coli. Synthetic purified CrArsM was crystallized in an unliganded form. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to space group R3:H, with unit-cell parameters a = b = 157.8, c = 95.4â Å, γ = 120° and two molecules in the asymmetric unit. Complete data sets were collected and processed to a resolution of 2.40â Å.