Conserved cysteine residues determine substrate specificity in a novel As(III) S‐adenosylmethionine methyltransferase from Aspergillus fumigatus

Methylation of inorganic arsenic is a central process in the organoarsenical biogeochemical cycle. Members of every kingdom have ArsM As(III) S‐adenosylmethionine (SAM) methyltransferases that methylates inorganic As(III) into mono‐ (MAs(III)), di‐ (DMAs(III)) and tri‐ (TMAs(III)) methylarsenicals. Every characterized ArsM to date has four conserved cysteine residues. All four cysteines are required for methylation of As(III) to MAs(III), but methylation of MAs(III) to DMAs(III) requires only the two cysteines closest to the C‐terminus. Fungi produce volatile and toxic arsines, but the physiological roles of arsenic methylation and the biochemical basis is unknown. Here they demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues. The genome of Aspergillus fumigatus has four arsM genes encoding ArsMs with only the second, third and fourth conserved cysteine residues. AfArsM1 methylates MAs(III) but not As(III). Heterologous expression of AfarsM1 in an Escherichia coli conferred resistance to MAs(III) but not As(III). The existence of ArsMs with only three conserved cysteine residues suggest that the ability to methylate MAs(III) may be an evolutionary step toward enzymes capable of methylating As(III), the result of a loss of function mutation in organisms with infrequent exposure to inorganic As(III) or as a resistance mechanism for MAs(III).     

High-throughput screening-compatible assays of As(III) S-adenosylmethionine methyltransferase activity

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.     

Identification of the catalytic nucleophile of tRNA (m5U54)methyltransferase

A covalent complex between tRNA (m5U54)methyltransferase, 5-fluorouridine tRNA(Phe), and S-adenosyl-L-[methyl-3H]methionine was formed in vitro and purified. Previously, it was shown that in this complex the 6-position of fluorouridine-54 is covalently linked to a catalytic nucleophile and the 5-position is bound to the transferred methyl group of AdoMet [Santi, D. V., & Hardy, L. W. (1987) Biochemistry 26, 8599-8606]. Proteolysis of the complex generated a [3H]methyl-FUtRNA-bound peptide, which was purified by 7 M urea-15% polyacrylamide gel electrophoresis. The peptide component of the complex was sequenced by gas-phase Edman degradation and found to contain two cysteines. The tritium was shown to be associated with Cys 324 of the methyltransferase, which unequivocally identifies this residue as the catalytic nucleophile.     

Arsenic methylation by a novel ArsM As(III) S‐adenosylmethionine methyltransferase that requires only two conserved cysteine residues

Arsenic (As) biomethylation is an important component of the As biogeochemical cycle that can influence As toxicity and mobility in the environment. Biomethylation of As is catalyzed by the enzyme arsenite (As[III]) S‐adenosylmethionine methyltransferase (ArsM). To date, all identified ArsM orthologs with As(III) methylation activities have four conserved cysteine residues, which are thought to be essential for As(III) methylation. Here, we isolated an As(III)‐methylating bacterium, Bacillus sp. CX‐1, and identified a gene encoding a S‐adenosylmethionine methyltranserase termed BlArsM with low sequence similarities (≤ 39%) to other ArsMs. BlArsM has six cysteine residues (Cys10, Cys11, Cys145, Cys193, Cys195 and Cys268), three of which (Cys10, Cys145 and Cys195) align with conserved cysteine residues found in most ArsMs. BlarsM is constitutively expressed in Bacillus sp. CX‐1. Heterologous expression of BlarsM conferred As(III) resistance. Purified BlArsM methylated both As(III) and methylarsenite (MAs[III]), with a final product of dimethylarsenate (DMAs[V]). When all six cysteines were individually altered to serine residues, only C145S and C195S derivatives lost the ability to methylate As(III) and MAs(III). The derivative C10S/C11S/C193S/C268S was still active. These results suggest that BlArsM is a novel As(III) S‐adenosylmethionine methyltransferase requiring only two conserved cysteine residues. A model of As(III) methylation by BlArsM is proposed.     

Equilibrium characterization of the As(III)-cysteine and the As(III)-glutathione systems in aqueous solution

Some arsenic compounds were the first antimicrobial agents specifically synthesized for the treatment of infectious diseases such as syphilis and trypanosomiasis. More recently, arsenic trioxide has been shown to be efficient in the treatment of acute promyelocytic leukemia. The exact mechanism of action has not been elucidated yet, but it seems to be related to arsenic binding to vicinal thiol groups of regulatory proteins. Glutathione is the major intracellular thiol and plays important roles in the cellular defense and metabolism. This paper reports on a study of the interactions between arsenic(III) and either cysteine or glutathione in aqueous solution. The behavior observed for the As(III)-glutathione system is very similar to that of As(III)-cysteine. In both cases, the formation of two complexes in aqueous solution was evidenced by NMR and electronic spectroscopies and by potentiometry. The formation constants of the cysteine complexes [As(H(-1)Cys)(3)], log K = 29.84(6), and [As(H(-2)Cys)(OH)(2)](-), log K = 12.01(9), and of the glutathione complexes [As(H(-2)GS)(3)](3-), log K = 32.0(6), and [As(H(-3)GS)(OH)(2)](2-), log K = 10(3) were calculated from potentiometric and spectroscopic data. In both cases, the [As(HL)(3)] species, in which the amine groups are protonated, predominate from acidic to neutral media, and the [As(L)(OH)(2)] species appear in basic medium (the charges were omitted for the sake of simplicity). Spectroscopic data clearly show that the arsenite-binding site in both complexes is the sulfur atom of cysteine. In the [As(L)(OH)(2)] species, the coordination sphere is completed by two hydroxyl groups. In both cases, arsenic probably adopts a trigonal pyramidal geometry. Above pH 10, the formation of [As(OH)(2)O](-) excludes the thiolates from arsenic coordination sites. At physiological pH, almost 80% of the ligand is present as [As(HL)(3)].     

Identification of functionally important residues of Arabidopsis thaliana S-adenosylmethionine decarboxylase

The Arabidopsis thaliana S-adenosylmethionine decarboxylase (AdoMetDC) cDNA (GenBank(TM) U63633) was cloned, and the AdoMetDC protein was expressed, purified, and characterized. The K(m) value for S-adenosylmethionine (AdoMet) is 23.1 microM and the K(i) value for methylglyoxal bis-(guanylhydrazone) (MGBG) is 0.15 microM. Site-specific mutagenesis was performed on the AdoMetDC to introduce mutations at conserved cysteine (Cys(50), Cys(83), and Cys(230)) and lysine(81) residues, chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. The AdoMetDC mutants K81A and C83A retained up to 60 and 10% of wild type activity, respectively, demonstrating that lysyl and sulfhydryl groups are required for full catalytic activity. However, changing Cys(50) and Cys(230) to alanine had minimal effects on the catalytic activity. Changing Lys(81) to alanine produced an altered substrate specificity. When lysine was used as a substrate instead of AdoMet, the substrate specificity for lysine increased 6-fold. The K(m) value for AdoMet is 11-fold higher than that of the wild type, but the V(max) value is more than 60%. Taken together, the results suggest that the lysine(81) residue is critical for substrate binding.     

ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase

The toxic metalloid arsenic is widely disseminated in the environment and causes a variety of health and environment problems. As an adaptation to arsenic-contaminated environments, organisms have developed resistance systems. Many ars operons contain only three genes, arsRBC. Five gene ars operons have two additional genes, arsD and arsA, and these two genes are usually adjacent to each other. ArsA from Escherichia coli plasmid R773 is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump. ArsD was recently identified as an arsenic chaperone to the ArsAB pump, transferring the trivalent metalloids As(III) and Sb(III) to the ArsA subunit of the pump. This increases the affinity of ArsA for As(III), resulting in increased rates if extrusion and resistance to environmentally relevant concentrations of arsenite. ArsD is a homodimer with three vicinal cysteine pairs, Cys12–Cys13, Cys112–Cys113 and Cys119–Cys120, in each subunit. Each vicinal pair binds one As(III) or Sb(III). ArsD mutants with alanines substituting for Cys112, Cys113, Cys119 or Cys120, individually or in pairs or truncations lacking the vicinal pairs, retained ability to interact with ArsA, to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys12, Cys13 or Cys18, individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. It is proposed that ArsD residues Cys12, Cys13 and Cys18, but not Cys112, Cys113, Cys119 or Cys120, are required for delivery of As(III) to and activation of the ArsAB pump.     

Novel inhibitors of As(III) S-adenosylmethionine methyltransferase (AS3MT) identified by virtual screening

Due to increased interest in As(III) S-adenosylmethionine methyltransferase (AS3MT), a search for chemical probes that can help elucidate function was initiated. A homology model was built based on related enzymes, and virtual screening produced 426 potential hits. Evaluation of these compounds in a functional enzymatic assay revealed several modest inhibitors including an O-substituted 2-amino-3-cyano indole scaffold. Two iterations of near neighbor searches revealed compound 5 as a potent inhibitor of AS3MT with good selectivity over representative methyltransferases DOT1L and NSD2 as well as a representative set of diverse receptors. Compound 5 should prove to be a useful tool to investigate the role of AS3MT and a potential starting point for further optimization.     

微生物氧化As(III)和Sb(III)的研究进展

砷(Arsenic,As)和锑(Antimony,Sb)属于同族元素,具有相似的化学性质,是公认的有毒类金属(metalloid),广泛存在于自然界中。随着人类的发展,环境中砷和锑的污染日益严重,类金属污染环境的修复已经刻不容缓。现已表明,自然界中的微生物在砷和锑的生物地球化学循环中发挥着重要的作用,尤其是微生物的氧化作用,可以将毒性较强的亚砷酸盐[Arsenite,As(III)]和亚锑酸盐[Antimonite,Sb(III)]氧化为毒性较低的砷酸盐[Arsenate,As(V)]和锑酸盐[Antimonate,Sb(V)],被认为是一种潜在的类金属污染环境修复方法。本文就国内外对As(III)氧化菌和Sb(III)氧化菌的多样性、As(III)和Sb(III)微生物氧化调控机制和应用的研究进展进行总结,旨在为深入了解和探索微生物介导的砷和锑生物地球化学循环及污染环境的微生物修复提供参考。     

Identification of the catalytic residues of AroA (Enolpyruvylshikimate 3-phosphate synthase) using partitioning analysis

AroA (EPSP synthase) catalyzes carboxyvinyl transfer through addition of shikimate 3-phosphate (S3P) to phosphoenolpyruvate (PEP) to form a tetrahedral intermediate (THI), followed by phosphate elimination to give enolpyruvylshikimate 3-phosphate (EPSP). A novel approach, partitioning analysis, was used to elucidate the roles of catalytic residues in each step of the reaction. Partitioning analysis involved trapping and purifying [1-(14)C]THI, degrading it with AroA, and quantitating the products. Wild-type AroA gave a partitioning factor, f(PEP) = 0.25 +/- 0.02 at pH 7.5, where f(PEP) = [[1-(14)C]PEP]/([[1-(14)C]PEP] + [[1-(14)C]EPSP]). Eighteen mutations were made to 14 amino acids to discover which residues preferentially catalyzed either the addition or the elimination step. Mutating a residue catalyzing one step (e.g., addition) should change f(PEP) to favor the opposite step (e.g., elimination). No mutants caused large changes in f(PEP), with experimental values from 0.07 to 0.41. This implied that there are no side chains that catalyze only addition or elimination, which further implied that the same residues are general acid/base catalysts in both forward and reverse THI breakdown. Only Lys22 (protonating S3P hydroxyl or phosphate) and Glu341 (deprotonating C3 of PEP) are correctly situated in the active site. In the overall reaction, Lys22 would act as a general base during addition, while Glu341 would act as a general acid. Almost half of the mutations (eight of 18) caused a >1000-fold decrease in specific activity, demonstrating that a large number of residues are important for transition state stabilization, "ensemble catalysis", in contrast to some enzymes where a single amino acid can be responsible for up to 10(8)-fold catalytic enhancement.     

Identification of Pyrococcus furiosus amylopullulanase catalytic residues

Pyrococcus furiosus amylopullulanase (PfAPU) belongs to glycosyl hydrolase family 57. Using sequence alignments of the known family 57 enzymes and site-directed mutagenesis, E291, D394, and E396 were identified as PfAPU putative catalytic residues. The apparent catalytic efficiencies (k_cat/K_m) of PfAPU mutants E291Q and D394N on pullulan were 123.0 and 24.4 times lower, respectively, than that of PfAPU. The activity of mutant E396Q on pullulan was too low to allow reliable determination of its catalytic efficiency. The apparent specific activities of these enzymes on starch also decreased 91.0 times (E291Q), 11.7 times (D394N), and 37.2 times (E396Q). The hydrolytic patterns for pullulan and starch were the same, while the hydrolysis rates differed as reported. Based on sequence alignment and a previous report, E291 is proposed as the catalytic nucleophile.     

Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm

The 16S rRNA methyltransferase Sgm from "Micromonospora zionensis" confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of the S-adenosyl-L-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with a K(D) (binding constant) of 17.6 microM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.     

Identification of Agrostis tenuis leaf proteins in response to As(V) and As(III) induced stress using a proteomics approach

Agrostis tenuis is known to be able to metabolise arsenate (As(V)) and arsenite (As(III)) which are toxic salts for most plants. A proteomic approach was developed to identify proteins expressed in response to treatments with these salts. A. tenuis plants were grown hydroponically in the presence of 134 and 668 μM As(V) or As(III) for 8 days at pH 7. During arsenic treatments, leaves showed chlorotic symptoms but fresh and dry leaf weights were not reduced, except in the presence of 668 μM As(III). On the contrary, a slight increase in biomass was observed with high As(V) concentrations. Thus, A. tenuis was more sensitive to As(III) than to As(V) and biomass was affected. Proteomic analysis enabled identification of a set of A. tenuis leaf proteins differentially expressed in response to arsenic exposure including a major functionally homogeneous group of enzymes such as oxygen-evolving enhancer protein, RuBisCO small and large subunits, RuBisCO activase and ATP synthase involved in the Calvin or Krebs cycle. The adaptative response to treatments resulted in partial disruption of the photosynthetic processes with prominent fragmentation of the RubisCO. Other proteins expressed differently from controls were identified and are possibly involved in the tolerance mechanisms of A. tenuis to arsenic treatments.     

Toxicokinetics and Biotransformation of As(III) and As(V) in Eisenia fetida

Earthworms, Eisenia fetida, were exposed to soils spiked with As(III) and As(V), to understand the response of earthworms to As in terms of both toxicity and accumulation using toxicokinetics, and to explain As metabolism and bioavailability. As(III) showed higher toxicity than As(V), in all toxicity endpoints of burrowing time, survival, growth, and cocoon production. Bioconcentration occurred at both As(III) and As(V) treatments during 28-d exposure. Worms did not show the elimination of As for consecutive 28 d of exposure to clean soils. Biotransformation of As was characterized using HPLC-ICP-MS and XANES, showing the reduction of As in worms regardless of the As species to which worms were exposed. Metabolism of As in worms that formed As-thiol complex is thought to limit the excretion of As, and thus induce bioconcentration in worms. Uptake rates by one-compartment model indicated that pore water was the bioaccessible pool of As, and directly controlled the uptake of As by worms. The study suggests that higher uptake rate and bioaccumulation of As(III) than of As(V) are among the factors that make As(III) more toxic than As(V).     

The ArsD As(III) metallochaperone

Arsenic, a toxic metalloid widely existing in the environment, causes a variety of health problems. The ars operon encoded by Escherichia coli plasmid R773 has arsD and arsA genes, where ArsA is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump, and ArsD is an arsenic chaperone for ArsA. ArsD transfers As(III) to ArsA and increases the affinity of ArsA for As(III), allowing resistance to environmental concentrations of arsenic. Cys12, Cys13 and Cys18 in ArsD form a three sulfur-coordinated As(III) binding site that is essential for metallochaperone activity. ATP hydrolysis by ArsA is required for transfer of As(III) from ArsD to ArsA, suggesting that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle. The 1.4 Å x-ray crystal structure of ArsD shows a core of four ??-strands flanked by four ??-helices in a thioredoxin fold. Docking of ArsD with ArsA was modeled in silico. Independently ArsD mutants exhibiting either weaker or stronger interaction with ArsA were selected. The locations of the mutations mapped on the surface of ArsD are consistent with the docking model. The results suggest that the interface with ArsA involves one surface of ??1 helix and metalloid binding site of ArsD.     

嗜酸热脂环酸杆菌中甘露聚糖酶活性位点的确立

【目的】通过定点突变确定嗜酸热脂环酸杆菌中甘露聚糖酶的活性催化位点。【方法】根据序列比对和GH53家族的结构信息选择可能的催化活性位点,利用重叠PCR法构建定点突变体,采用薄层层析(TLC)法和3,5-二硝基水杨酸(DNS)法检测各酶蛋白活性。【结果】通过重叠PCR法成功构建了7个位点的突变体,其中第150和159位的氨基酸突变对活性改变甚少或几乎没有,而第151和231位谷氨酸的羧基基团的改变以及双位点突变体E2Q则导致其对各种底物催化活性的丧失,说明位于β4和β7折叠的C末端的E151和E231的羧基基团作为功能基团参与了催化反应。【结论】E151和E231分别是新型甘露聚糖酶AaManA的酸碱催化位点和亲核催化位点。     

Identification of the catalytic residues of bifunctional glycogen debranching enzyme

Eukaryotic glycogen debranching enzyme (GDE) possesses two different catalytic activities (oligo-1,4-->1,4-glucantransferase/amylo-1,6-glucosidase) on a single polypeptide chain. To elucidate the structure-function relationship of GDE, the catalytic residues of yeast GDE were determined by site-directed mutagenesis. Asp-535, Glu-564, and Asp-670 on the N-terminal half and Asp-1086 and Asp-1147 on the C-terminal half were chosen by the multiple sequence alignment or the comparison of hydrophobic cluster architectures among related enzymes. The five mutant enzymes, D535N, E564Q, D670N, D1086N, and D1147N were constructed. The mutant enzymes showed the same purification profiles as that of wild-type enzyme on beta-CD-Sepharose-6B affinity chromatography. All the mutant enzymes possessed either transferase activity or glucosidase activity. Three mutants, D535N, E564Q, and D670N, lost transferase activity but retained glucosidase activity. In contrast, D1086N and D1147N lost glucosidase activity but retained transferase activity. Furthermore, the kinetic parameters of each mutant enzyme exhibiting either the glucosidase activity or transferase activity did not vary markedly from the activities exhibited by the wild-type enzyme. These results strongly indicate that the two activities of GDE, transferase and glucosidase, are independent and located at different sites on the polypeptide chain.     

Identification of the catalytic residues in the double-zinc aminopeptidase from Streptomyces griseus

The aminopeptidase from Streptomyces griseus (SGAP) has been cloned and expressed in Escherichia coli. By growing the cells in the presence of 1 M sorbitol at 18 degrees C, the protein was obtained in a soluble and active form. The amino acid sequence of the recombinant SGAP contained four amino acids differing from the previously published sequence. Re-sequencing of the native protein indicated that asparagines 70 and 184 are in fact aspartic acids as in the recombinant protein. Based on the crystal structure of SGAP, Glu131 and Tyr246 were proposed to be the catalytic residues. Replacements of Glu131 resulted in loss of activity of 4-5 orders of magnitude, consistent with Glu131 acting as the general base residue. Mutations in Tyr246 resulted in about 100-fold reduction of activity, suggesting that this residue is involved in the stabilization of the transition state intermediate.     

Metabolism of S-adenosylmethionine in rat hepatocytes: transfer of methyl group from S-adenosylmethionine by methyltransferase reactions

Treatment of rats with a methionine diet leads not only to a marked increase of S-adenosylmethionine synthetase in liver, but also to the increase of glycine, guanidoacetate and betaine-homocysteine methyltransferases. The activity of tRNA methyltransferase decreased with the increased amounts of methionine in the diets. However, the activities of phospholipids and S-adenosylmethionine-homocysteine methyltransferases did not show any significant change. When hepatocarcinogenesis induced by 2-fluorenylacetamide progresses, the activities of glycine and guanidoacetate methyltransferases in rat liver decreased, and could not be detected in tumorous area 8 months after treatment. The levels of S-adenosylmethionine in the liver also decreased to levels of one-fifth of control animals at 8 months. The uptake and metabolism of [methyl-3H]-methionine and -S-adenosylmethionine have been investigated by in vivo and isolated hepatocytes. The uptake of methionine and transfer of methyl group to phospholipid in the cells by methionine were remarkably higher than those by S-adenosylmethionine. These results indicate that phospholipids in hepatocytes accept methyl group from S-adenosylmethionine immediately, when it is synthesized from methionine, before mixing its pool in the cells.     

Identification of catalytic residues in glyoxal oxidase by targeted mutagenesis

Glyoxal oxidase is a copper metalloenzyme produced by the wood-rot fungus Phanerochaete chrysosporium as an essential component of its extracellular lignin degradation pathways. Previous spectroscopic studies on glyoxal oxidase have demonstrated that it contains a free radical-coupled copper active site remarkably similar to that found in another fungal metalloenzyme, galactose oxidase. Alignment of primary structures has allowed four catalytic residues of glyoxal oxidase to be targeted for site-directed mutagenesis in the recombinant protein. Three glyoxal oxidase mutants have been heterologously expressed in both a filamentous fungus (Aspergillus nidulans) and in a methylotrophic yeast (Pichia pastoris), the latter expression system producing as much as 2 g of protein per liter of culture medium under conditions of high density methanol-induced fermentation. Biochemical and spectroscopic characterization of the mutant enzymes supports structural correlations between galactose oxidase and glyoxal oxidase, clearly identifying the catalytically important residues in glyoxal oxidase and demonstrating the functions of each of these residues.