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1.
    
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 = 95.4 Å, γ = 120° and two molecules in the asymmetric unit. Complete data sets were collected and processed to a resolution of 2.40 Å.  相似文献   

2.
    
Methylation of the toxic metalloid arsenic is widespread in nature. Members of every kingdom have arsenic(III) S‐adenosylmethionine (SAM) methyltransferase enzymes, which are termed ArsM in microbes and AS3MT in animals, including humans. Trivalent arsenic(III) is methylated up to three times to form methylarsenite [MAs(III)], dimethylarsenite [DMAs(III)] and the volatile trimethylarsine [TMAs(III)]. In microbes, arsenic methylation is a detoxification process. In humans, MAs(III) and DMAs(III) are more toxic and carcinogenic than either inorganic arsenate or arsenite. Here, new crystal structures are reported of ArsM from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508 (CmArsM) with the bound aromatic arsenicals phenylarsenite [PhAs(III)] at 1.80 Å resolution and reduced roxarsone [Rox(III)] at 2.25 Å resolution. These organoarsenicals are bound to two of four conserved cysteine residues: Cys174 and Cys224. The electron density extends the structure to include a newly identified conserved cysteine residue, Cys44, which is disulfide‐bonded to the fourth conserved cysteine residue, Cys72. A second disulfide bond between Cys72 and Cys174 had been observed previously in a structure with bound SAM. The loop containing Cys44 and Cys72 shifts by nearly 6.5 Å in the arsenic(III)‐bound structures compared with the SAM‐bound structure, which suggests that this movement leads to formation of the Cys72–Cys174 disulfide bond. A model is proposed for the catalytic mechanism of arsenic(III) SAM methyltransferases in which a disulfide‐bond cascade maintains the products in the trivalent state.  相似文献   

3.
    
Wheat‐germ protein l ‐isoaspartyl O‐methyltransferase (WPIMT) can initiate the conversion of l ‐isoaspartyl residues in a protein or peptide, which accumulate during the aging process in wheat‐germ seeds, to normal l ‐aspartyl groups. The recombinant protein of WPIMT was overexpressed in Escherichia coli and purified to homogeneity. The protein was crystallized in the presence of S‐­adenosine‐l ‐homocysteine using 2‐methyl‐2,4‐pentanediol. Preliminary X‐ray analysis indicated a tetragonal space group P41212 or P43212, with unit‐cell parameters a = b = 77.3, c = 152.9 Å for cryofrozen crystals at 90 K. The crystals diffracted to 3.3 Å and contain two molecules per asymmetric unit.  相似文献   

4.
    
Prokaryotic 5′‐methylthioadenosine/S‐adenosylhomocysteine nucleosidase (MtaN) is a multifunctional enzyme that can hydrolyze S‐adenosyl‐L‐homocysteine (SAH) and S‐methyl‐5′‐thioadenosine (MTA) to give S‐ribosyl‐L‐homocysteine (SRH) and S‐methyl‐5′‐thioribose (MTR), respectively. This reaction plays a key role in several metabolic pathways, including biological methylation, polyamine biosynthesis, methionine recycling and bacterial quorum sensing. Structurally, MtaN belongs to the MtnN subfamily of the purine nucleoside phosphorylase (PNP)/uridine phosphorylase (UDP) phosphorylase family. Aeromonas hydrophila has two MtnN subfamily proteins: MtaN‐1, a periplasmic protein with an N‐terminal signal sequence, and MtaN‐2, a cytosolic protein. In this study, MtaN‐1 from Aeromonas hydrophila was successfully expressed and purified using Ni–NTA affinity, Q anion‐exchange and gel‐filtration chromatography. Crystals of the protein in complex with the substrate SAH were obtained and diffracted to a resolution of 1.4 Å. The crystals belonged to the trigonal space group P3121 or P3221, with unit‐cell parameters a = b = 102.7, c = 118.8 Å. The asymmetric unit contained two molecules of MtaN‐1 complexed with SAH.  相似文献   

5.
    
Pyocyanin, phenazine‐1‐carboxylic acid and more than 70 related compounds collectively known as phenazines are produced by various species of Pseudomonas, including the fluorescent pseudomonad P. aeruginosa, a Gram‐negative opportunistic pathogen in humans and animals. P. aeruginosa synthesizes a characteristic blue water‐soluble compound called pyocyanin (1‐­hydroxy‐5‐methyl‐phenazine). Two enzymes designated PhzM and PhzS are involved in the terminal steps of its synthesis and very little is known about these enzymes. In this study, PhzM, a dimeric S‐adenosylmethionine‐dependent methyltransferase, was purified and crystallized from PEG 3350/sodium cacodylate/sodium citrate pH 6.5. The crystals belong to space group P1, with unit‐cell parameters a = 46.1, b = 61.8, c = 69.6 Å, α = 96.3, β = 106.6, γ = 106.9°. They contain one dimer in the asymmetric unit and diffract to a resolution of 1.8 Å. Anomalous data to 2.3 Å resolution have been collected from seleno‐l ‐­methionine‐labelled PhzM.  相似文献   

6.
    
The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S‐adenosyl‐l ‐methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino‐acid sequence motifs common to class I Rossmann‐fold‐like MTases suggested a specific role as an RNA 5‐methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet‐labelled apoprotein were obtained: SeMet‐ApoI and SeMet‐ApoII. Cocrystallization of the native protein with S‐­adenosyl‐l ‐homocysteine (AdoHcy) yielded a third crystal form, Native‐AdoHcy. The SeMet‐ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 Å resolution. The SeMet‐ApoII and Native‐AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 Å, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three‐domain structure composed of a small N‐terminal PUA domain, a central α/β‐domain and a C‐terminal Rossmann‐fold‐like MTase domain. The three domains form an overall clamp‐like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5‐carbon position. The cleft is suitable in size and charge distribution for binding single‐stranded RNA.  相似文献   

7.
    
Methionine adenosyltransferase (MAT) deficiency, characterized by isolated persistent hypermethioninemia (IPH), is caused by mutations in the MAT1A gene encoding MATαl, one of the major hepatic enzymes. Most of the associated hypermethioninemic conditions are inherited as autosomal recessive traits; however, dominant inheritance of hypermethioninemia is caused by an Arg264His (R264H) mutation. This mutation has been confirmed in a screening programme of newborns as the most common mutation in babies with IPH. Arg264 makes an inter‐subunit salt bridge located at the dimer interface where the active site assembles. Here, it is demonstrated that the R264H mutation results in greatly reduced MAT activity, while retaining its ability to dimerize, indicating that the lower activity arises from alteration at the active site. The first crystallographic structure of the apo form of the wild‐type MATαl enzyme is provided, which shows a tetrameric assembly in which two compact dimers combine to form a catalytic tetramer. In contrast, the crystal structure of the MATαl R264H mutant reveals a weaker dimeric assembly, suggesting that the mutation lowers the affinity for dimer–dimer interaction. The formation of a hetero‐oligomer with the regulatory MATβV1 subunit or incubation with a quinolone‐based compound (SCR0911) results in the near‐full recovery of the enzymatic activity of the pathogenic mutation R264H, opening a clear avenue for a therapeutic solution based on chemical interventions that help to correct the defect of the enzyme in its ability to metabolize methionine.  相似文献   

8.
    
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9.
    
Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well‐studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S‐adenosylmethionine and a short‐chain polyamine (putrescine) to make a medium‐chain polyamine (spermidine) and 5′‐deoxy‐5′‐methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S‐adenosylhomocysteine (dcSAH). The inhibitor was synthesized, and dose‐dependent inhibition of human, Thermatoga maritima, and Plasmodium falciparum spermidine synthases, as well as functionally homologous human spermine synthase, was determined. The human SpdS/dcSAH complex structure was determined by X‐ray crystallography at 2.0 Å resolution and showed consistent active site positioning and coordination with previously known structures. Isothermal calorimetry binding assays confirmed inhibitor binding to human SpdS with Kd of 1.1 ± 0.3 μM in the absence of putrescine and 3.2 ± 0.1 μM in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold.  相似文献   

10.
    
A cDNA from the plant Thalictrum flavum encoding pavine N‐methyltransferase, an enzyme belonging to a novel class of S‐adenosylmethionine‐dependent N‐methyltransferases specific for benzylisoquinoline alkaloids, has been heterologously expressed in Escherichia coli. The enzyme was purified using affinity and gel‐filtration chromatography and was crystallized in space group P21. The structure was solved at 2.0 Å resolution using a xenon derivative and the single isomorphous replacement with anomalous scattering method.  相似文献   

11.
    
The expression of LeATL6, which encodes RING‐H2 zinc finger ubiquitin‐protein ligase E3, is highly induced in tomato roots treated with the elicitin‐like cell wall protein fraction (CWP) from the non‐pathogenic oomycete Pythium oligandrum, which enhances resistance to pathogens through a jasmonic acid (JA)‐dependent signalling pathway. In this study, the role of LeATL6 for CWP‐induced defence response was further analysed. To screen the putative target protein of LeATL6 for the CWP‐induced defence mechanism in tomato, we used a yeast two‐hybrid system to screen five clones encoding a protein that interacts with LeATL6. Four clones had a function associated with the ubiquitin‐proteasome system. Another positive clone encoded a protein sharing homology with S‐adenosylmethionine decarboxylase (SAMDC). In CWP‐treated tomato roots, SAMDC activity was clearly suppressed. Thus, the interaction of SAMDC with LeATL6 and the decreased SAMDC activity may be associated with JA‐dependent induced resistance in tomato treated with P. oligandrum.  相似文献   

12.
    
N‐Carbamoyl‐l ‐amino‐acid amidohydrolases (l ‐N‐carbamoylases; EC 3.5.1.87) hydrolyze the carbon–nitrogen bond of the ureido group in N‐carbamoyl‐l ‐α‐amino acids. These enzymes are commonly used in the production of optically pure natural and non‐natural l ‐amino acids using the `hydantoinase process'. Recombinant l ‐N‐carbamoylase from Geobacillus stearothermophilus CECT43 has been expressed, purified and crystallized by hanging‐drop vapour diffusion. X‐ray data were collected to a resolution of 2.75 Å. The crystals belonged to space group P21212, with unit‐cell parameters a = 103.2, b = 211.7, c = 43.1 Å and two subunits in the asymmetric unit.  相似文献   

13.
Xanthomonas campestris pv. campestris (Xcc) causes black rot, one of the most important diseases of brassica crops worldwide. The type III effector inventory plays important roles in the virulence and pathogenicity of the pathogen. However, little is known about the virulence function(s) of the putative type III effector AvrXccB in Xcc. Here, we investigated the immune suppression ability of AvrXccB and the possible underlying mechanisms. AvrXccB was demonstrated to be secreted in a type III secretion system‐dependent manner. AvrXccB tagged with green fluorescent protein is localized to the plasma membrane in Arabidopsis, and the putative N‐myristoylation motif is essential for its localization. Chemical‐induced expression of AvrXccB suppresses flg22‐triggered callose deposition and the oxidative burst, and promotes the in planta growth of Xcc and Pseudomonas syringae pv. tomato in transgenic Arabidopsis plants. The putative catalytic triad and plasma membrane localization of AvrXccB are required for its immunosuppressive activity. Furthermore, it was demonstrated that AvrXccB interacts with the Arabidopsis S‐adenosyl‐l ‐methionine‐dependent methyltransferases SAM‐MT1 and SAM‐MT2. Interestingly, SAM‐MT1 is not only self‐associated, but also associated with SAM‐MT2 in vivo. SAM‐MT1 and SAM‐MT2 expression is significantly induced upon stimulation of microbe‐associated molecular patterns and bacterial infection. Collectively, these findings indicate that AvrXccB targets a putative methyltransferase complex and suppresses plant immunity.  相似文献   

14.
    
The genes encoding the tRNA‐modifying enzyme S‐adenosylmethionine:tRNA ribosyl transferase/isomerase (QueA) from 12 eubacterial sources were overexpressed in Escherichia coli and the resulting products were purified to homogeneity and subjected to crystallization trials. Using the hanging‐drop vapour‐diffusion method, crystals suitable for X‐ray diffraction experiments were only obtained for the queA gene product from Bacillus subtilis. The crystals belong to the space group P422, with unit‐cell parameters a = b = 100.7, c = 150.9 Å. Using highly focused synchrotron radiation from the EMBL/ESRF beamline ID13 (Grenoble, France), diffraction to at least 3.2 Å could be achieved. A selenomethionyl derivative of the protein was prepared and crystallized for future multiwavelength anomalous diffraction (MAD) experiments.  相似文献   

15.
    
Nosiheptide‐resistance methyltransferase (NSR) methylates 23S rRNA at the nucleotide adenosine 1067 in Escherichia coli and thus contributes to resistance against nosiheptide, a sulfur‐containing peptide antibiotic. Here, the expression, purification and crystallization of NSR from Streptomyces actuosus are reported. Diffracting crystals were grown by the hanging‐drop vapour‐diffusion method in reservoir solution consisting of 0.35 M ammonium chloride, 24%(w/v) PEG 3350, 0.1 M MES pH 5.7 at 293 K. Native data have been collected from the apo enzyme and a SAM complex, as well as apo SeMet SAD data. The diffraction patterns of the apo form of NSR, of NSR complexed with SAM and of SeMet‐labelled NSR crystals extended to 1.90, 1.95 and 2.25 Å resolution, respectively, using synchrotron radiation. All crystals belonged to space group P21, with approximate unit‐cell parameters a = 64.6, b = 69.6, c = 64.9 Å, β = 117.8°.  相似文献   

16.
    
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17.
    
A novel N‐acetyl‐l ‐citrulline deacetylase that is able to catalyze the hydrolysis of N‐acetyl‐l ‐citrulline to acetate and citrulline was identified from Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the monoclinic space group C2 and diffract to 1.75 Å resolution, with unit‐cell parameters a = 94.13, b = 95.23, c = 43.61 Å, β = 93.76°. Since attempts to use homologous structural models to solve the structure via molecular replacement were unsuccessful, the selenomethionine‐substituted protein was prepared using an overnight auto‐induction overexpression system. Selenomethionine incorporation into the protein was verified by MALDI–TOF/TOF mass‐spectroscopic analysis after trypsin digestion. The crystals of the selenomethionine‐substituted protein were prepared using crystallization conditions similar to those for the native protein. Multiple anomalous dispersion (MAD) data were collected at Brookhaven National Laboratory. Structure determination is under way using the MAD phasing method.  相似文献   

18.
    
S‐Adenosylmethionine (AdoMet) participates in a wide range of methylation and other group‐transfer reactions and also serves as the precursor for two groups of quorum‐sensing molecules that function as regulators of the production of virulence factors in Gram‐negative bacteria. The synthesis of AdoMet is catalyzed by AdoMet synthetases (MATs), a ubiquitous family of enzymes found in species ranging from microorganisms to mammals. The AdoMet synthetase from the bacterium Campylobacter jejuni (cjMAT) is an outlier among this homologous enzyme family, with lower sequence identity, numerous insertions and substitutions, and higher catalytic activity compared with other bacterial MATs. Alterations in the structure of this enzyme provide an explanation for its unusual dimeric quaternary structure relative to the other MATs. Taken together with several active‐site substitutions, this new structure provides insights into its improved kinetic properties with alternative substrates.  相似文献   

19.
    
1‐Methyladenine (1‐MeAde), the oocyte maturation‐inducing substance of starfish, is produced by ovarian follicle cells upon stimulation with a gonad‐stimulating substance (GSS) released from radial nerves. It has been reported that a process of methylation is involved in GSS‐induced 1‐MeAde production by starfish ovarian follicle cells. The present study sought to identify a possible methyl donor for 1‐MeAde biosynthesis in follicle cells of the starfish Asterina pectinifera. When isolated follicle cells were incubated with [methyl‐14C]methionine (Met), there was an increase in the level of radiolabeled S‐adenosylmethionine (SAM). After further incubation with GSS, the [methyl‐14C]SAM level decreased, concomitant with a marked increase in radiolabeled 1‐MeAde production. The amount of [methyl‐14C]SAM consumed under the influence of GSS was similar to the amount of [methyl‐14C]1‐MeAde produced. Therefore, it is concluded that SAM is a methyl donor for 1‐MeAde biosynthesis in starfish ovarian follicle cells. On the other hand, it is likely that the purine molecule of 1‐MeAde is not derived from SAM but from ATP. 3‐Isobutyl‐1‐methylxanthine, a potent inhibitor of cyclic AMP phosphodiesterase, also caused a reduction in the level of radiolabeled SAM following 1‐MeAde production. This suggests that the methylation process of 1‐MeAde biosynthesis in starfish ovarian follicle cells upon stimulation with GSS is mediated by a second messenger, cyclic AMP. Mol. Reprod. Dev. 54:63–68, 1999. © 1999 Wiley‐Liss, Inc.  相似文献   

20.
    
S‐Adenosyl‐l ‐homocysteine hydrolase (SAHH; EC 3.3.1.1) catalyzes the reversible hydrolysis of S‐adenosyl‐l ‐homocysteine to adenosine and l ‐homocysteine. For crystallographic investigations, mouse SAHH (MmSAHH) was overexpressed in bacterial cells and crystallized using the hanging‐drop vapour‐diffusion method in the presence of the reaction product adenosine. X‐ray diffraction data to 1.55 Å resolution were collected from an orthorhombic crystal form belonging to space group I222 with unit‐cell parameters a = 100.64, b = 104.44, c = 177.31 Å. Structural analysis by molecular replacement is in progress.  相似文献   

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