共查询到20条相似文献,搜索用时 31 毫秒
1.
In humans, the enzyme thiopurine methyltransferase (TPMT) metabolizes 6-thiopurine (6-TP) medications, commonly used for immune suppression and for the treatment of hematopoietic malignancies. Genetic polymorphisms in the TPMT protein sequence accelerate intracellular degradation of the enzyme through an ubiquitylation and proteasomal-dependent pathway. Research has led to the hypothesis that these polymorphisms destabilize the native structure of TPMT, resulting in the formation of misfolded or partially unfolded states, which are subsequently recognized for intracellular degradation. Addition of the cosubstrate, S-adenosylmethionine (SAM), prevents degradation of the TPMT polymorphs in experimental assays, presumably by stabilizing the native structure. Using a bacterial orthologue of TPMT from Pseudomonas syringae, we have used NMR spectroscopy to describe the consequences of binding sinefungin, a SAM analogue, on the structure and dynamics of the TPMT protein backbone. NMR chemical shift mapping experiments localize sinefungin to a highly conserved site in classical methyltransferases. Distal chemical shift changes involving the presumed active site cover imply indirect conformational changes induced by sinefungin, which may play a role in substrate recognition or the catalytic mechanism. Analysis of protein backbone dynamics based on NMR relaxation reveals a combination of complementary effects. Whereas the peripheral, inserted structural elements of the TPMT topology are conformationally stabilized by the presence of sinefungin, a consistent increase in backbone mobility is observed for the central, conserved structural elements. The potential implications for the structural and dynamic effects of binding sinefungin for the catalytic mechanism of the enzyme and the stabilization of the degradation-susceptible TPMT polymorphs are discussed. 相似文献
2.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,674(2):247-252
A reversed-phase high-performance liquid chromatographic method for the determination of sinefungin, a new antiprotozoal drug, in rat plasma has been developed and validated. Sample preparation was performed at 4°C by deproteinization with acetonitrile. Vidarabine was used as an internal standard. Both sinefungin and vidarabine were separated on a C18 column with a mobile phase of ammmonium dihydrogenphosphate-acetonitrile (95:5, v/v) and detected by ultraviolet absorbance at 260 nm. Recoveries of sinefungin from plasma were 75 ± 3.2% and 81 ± 4.8% following dosage at concentrations of 10 μg/ml and 30 μ/ml, respectively. Using 25- μl of rat plasma the limit of quantitation was 1 μg/ml sinefungin, and the assay was linear from 1 to 30 μg/ml. This method appears sensitive enough to be used in further pharmacokinetic studies of sinefungin in animal models. 相似文献
3.
Sinefungin resistance of Saccharomyces cerevisiae arising from Sam3 mutations that inactivate the AdoMet transporter or from increased expression of AdoMet synthase plus mRNA cap guanine-N7 methyltransferase
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The S-adenosylmethionine (AdoMet) analog sinefungin is a natural product antibiotic that inhibits nucleic acid methyltransferases and arrests the growth of unicellular eukarya and eukaryal viruses. The basis for the particular sensitivity of fungi and protozoa to sinefungin is not known. Here we report the isolation and characterization of spontaneous sinefungin-resistant mutants of the budding yeast Saccharomyces cerevisiae. In all cases, sinefungin resistance was attributable to a loss-of-function mutation in Sam3, the yeast high-affinity AdoMet transporter. Overexpression of wild-type Sam3 increased the sensitivity of yeast to growth inhibition by sinefungin. Thus, Sam3 is a tunable determinant of sinefungin potency. The shared ability of protozoan parasites to import AdoMet might determine sinefungin's anti-infective spectrum. Insights to the intracellular action of sinefungin stem from the finding that increased gene dosage of yeast AdoMet synthase plus cap guanine-N7 methyltransferase afforded greater resistance to sinefungin than either enzyme alone. These results are consistent with the proposal that mRNA cap methylation is a principal target of sinefungin's bioactivity. 相似文献
4.
Structural analogs of S-adenosylhomocysteine were tested in vitro for inhibition of the yeast S-adenosylmethionine:delta 24-sterol-C-methyltransferase enzyme. A wide inhibitory range by these compounds was observed, suggesting which structural features of the parent compound are important for binding to the enzyme. No analog tested had inhibitory activity specific only for this enzyme. The most active compound was sinefungin, a metabolite of Streptomyces griseolus, which was also able to inhibit growth of yeast cultures. Sterol extracts of cells grown in the presence of sinefungin revealed a dramatic increase in the levels of zymosterol, the sterol substrate in the transmethylation under study, and a concomitant decrease in the levels of ergosterol. Evidence is presented that sinefungin is transported inside the cell by the same permease as S-adenosylmethionine. We conclude that sinefungin is blocking the in vivo methylation of sterols in yeast. The implications of this finding are discussed. 相似文献
5.
Regulation of sinefungin biosynthesis by the wild-type strain and mutants of Streptomyces incarnatus
Sinefungin, an antifungal and antiparasitic antibiotic, is produced efficiently from ammonium citrate by prototrophic strains of Streptomyces incarnatus. The regulation of the biosynthesis of this nucleoside, composed of adenosine and ornithine, was studied by using auxotrophic mutants and a resting-cell system. Mutants blocked in arginine synthesis were not able to produce sinefungin. A uridine-negative mutant produced sinefungin in the presence of ATP, but this production was strongly inhibited when amino acids of the urea cycle were added. The same mutant produced sinefungin from aspartic acid, and this production was enhanced by ornithine. Our results show that the ornithine part of the molecule originates from arginine, liberated by either anabolic or catabolic processes. 相似文献
6.
Raymond Cooper Pradip Das Claire Federbush Ronald Mierzwa Mahesh Patel Birendra Pramanik Imbi Truumees 《Journal of industrial microbiology & biotechnology》1990,5(1):1-8
Summary Characterization of sinefungin related antifungal antibiotics from fermentation broth was accomplished by coupling photodiode array (PDA) detection to high performance liquid chromatography (HPLC). From the combined HPLC-PDA evaluation of broth filtrate, we detected five sinefungin related components. Fast atom bombardment (FAB) mass spectroscopic evaluations, mass-analysed ion kinetic energy spectra (MIKES) and collision activated (CA) MIKES of these components confirmed their respective identities. Our findings from the combination of HPLC photodiode array acquisition and FAB-mass spectrometry suggest we have detected the presence of a previously unreported sinefungin analogue. 相似文献
7.
Regulation of sinefungin biosynthesis by the wild-type strain and mutants of Streptomyces incarnatus. 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Sinefungin, an antifungal and antiparasitic antibiotic, is produced efficiently from ammonium citrate by prototrophic strains of Streptomyces incarnatus. The regulation of the biosynthesis of this nucleoside, composed of adenosine and ornithine, was studied by using auxotrophic mutants and a resting-cell system. Mutants blocked in arginine synthesis were not able to produce sinefungin. A uridine-negative mutant produced sinefungin in the presence of ATP, but this production was strongly inhibited when amino acids of the urea cycle were added. The same mutant produced sinefungin from aspartic acid, and this production was enhanced by ornithine. Our results show that the ornithine part of the molecule originates from arginine, liberated by either anabolic or catabolic processes. 相似文献
8.
Zylicz-Stachula A Zołnierkiewicz O Jeżewska-Frąckowiak J Skowron PM 《BioTechniques》2011,50(6):397-406
The type IIS/IIC restriction endonuclease TspGWI recognizes the sequence 5'-ACGGA-3', cleaving DNA 11/9 nucleotides downstream. Here we show that sinefungin, a cofactor analog of S-adenosyl methionine, induces a unique type of relaxation in DNA recognition specificity. In the presence of sinefungin, TspGWI recognizes and cleaves at least 12 degenerate variants of the original recognition sequence that vary by single base pair changes from the original 5-bp restriction site with only a single degeneracy per variant appearing to be allowed. In addition, sinefungin was found to have a stimulatory effect on cleavage at these nondegenerate TspGWI recognition sites, irrespective of their number or the DNA topology. Interestingly, no fixed "core" could be identified among the new recognition sequences. Theoretically, TspGWI cleaves DNA every 1024 bp, while sinefungin-induced activity cleaves every 78.8 bp, corresponding to a putative 3-bp long recognition site. Thus, the combination of sinefungin and TspGWI represents a novel frequent cutter, next only to CviJI/CviJI*, that should prove useful in DNA cloning methodologies. 相似文献
9.
Stefan Berking 《Differentiation; research in biological diversity》1986,32(1):10-16
Abstract. The control of pattern formation, cell differentiation and cell proliferation in hydroids involves inhibitory signals. In an attempt to identify their chemical nature, compounds from coelenterates which interfere with metamorphosis and pattern-forming processes in Hydractinia and Eirene were isolated. The most strongly metamorphosis-inhibiting compounds were determined to be N -methylpicolinic acid (homarine), N -methylnicotinic acid (trigonelline) and N -trimethylglycine (betaine). The overal concentration of these compounds within tissues is in the range of several millimoles, but micromolar quantities were found to affect development. Thus, the substances must be mainly present in a stored or an inactivated form. The compounds appear to exert their influence by transfering methyl groups to as yet unknown targets. Chemically related compounds that are not able to function as methyl donors have no or only a much lower inhibitory influence, while potential methyl or ethyl donors such as methionine and ethionine have a strong inhibitory influence. Cycloleucine, a competitor with methionine in the production of S-adenosyl-methionine (SAM), and sinefungin, a competitor with SAM in transmethylation, interfere with the intrinsic morphogenetically active compounds identified. One of the spatial patterns controlled by inhibitory signals is the distance between polyps in colonies. In Eirene , the addition of N -methylpicolinic acid led to an increase in the interpolyp distance, while sinefungin produced a decrease in this distance. The addition of sinefungin also stimulated stolon branching. Thus, control of methylation appears to play a key role in the control of metamorphosis and pattern formation in hydrozoa. 相似文献
10.
Zheng S Hausmann S Liu Q Ghosh A Schwer B Lima CD Shuman S 《The Journal of biological chemistry》2006,281(47):35904-35913
Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo,14 reduced activity in vitro to < or = 2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin. 相似文献
11.
Lamya Moulay Malka Robert-Gero Spencer Brown Marie-Claude Gendron Frédéric Tournier 《Experimental cell research》1996,226(2):283
Sinefungin is an antibiotic possessing a strong anti-leishmanial activity. Among the most important effects of this molecule onLeishmania donovanipromastigotes are morphological modifications and a very rapid and effective inhibition of DNA synthesis. These cells contain a single DNA-rich mitochondrion whose division cycle is coordinated with the nuclear division cycle. We have developed a flow-cytometric procedure based upon mithramycin as fluorochrome that can perform quantitative cell cycle analysis on the nuclear DNA. Cell cycle progression was analyzed to establish that sinefungin irreversibly blocks the promastigotes in early S phase. Sinefungin did not react with stationary cells as they were arrested in G1. Surprisingly, taxol, a microtubule-stabilizing drug, induced the same morphological modifications as sinefungin although it interfered with the G2/M progression. According to immunofluorescence studies, the stable microtubular network is apparently affected neither by taxol nor by sinefungin. 相似文献
12.
13.
Adilson D. Da Silva Edmilson JoseMaria Pierre Blanchard Jean-Louis Fourrey Malka Robert-Gero 《Nucleosides, nucleotides & nucleic acids》2013,32(12):2175-2187
Abstract An approach to an asymmetric synthesis of carbocyclic sinefungin (cSF) 2 is proposed. The sequence, which uses an original radical based chemistry for C-C bond formation, led to the immediate precursor 18 of the protected desired compound. While the overall yield is modest, it is noticeable that only a limited number of steps are needed to obtain the target compound. 相似文献
14.
C. Barbés J. Sánchez M.J. Yebra M. Robert-Geró C. Hardisson 《FEMS microbiology letters》1990,69(3):239-243
Sinefungin is a naturally occurring nucleoside isolated from cultures of Streptomyces griseolus and S. incarnatus. It is structurally related to S-adenosyl-methionine (SAM) and S-adenosyl-L-homocysteine (SAH). Its effect and level of action on prokaryotes has not been studied with the same detail as with eukaryotic cells. In this report we describe the effect of sinefungin and SAH on several Streptomyces methyltransferases (DNA and protein MTases) and on other bacterial DNA-MTases. Protein MTases are resistant to sinefungin, whereas DNA-MTases are inhibited. Adenine MTases however, seem more sensitive to this analogue than cytosine MTases. 相似文献
15.
Protein methylases in Trypanosoma brucei brucei: activities and response to DL-alpha-difluoromethylornithine 总被引:1,自引:0,他引:1
Protein methylases I, II and III were detected in extracts of Trypanosoma brucei brucei, and characterized according to the specific amino substituent methylated. Only protein methylase II activity was elevated by difluoromethylornithine treatment of T. b. brucei, and hence this enzyme was characterized further. Protein methylase II transferred methyl groups from S-adenosyl-L-methionine (S-AdoMet) to the carboxyl residues of several protein substrates, exhibiting highest activity with histone VIII-S (arginine-rich subgroup f3). The crude enzyme had an apparent Km for histone VIII-S of 28 mg ml-1 (11.4 mM-aspartyl and 18.4 mM-glutamyl residues methylated), and an apparent Km for S-AdoMet of 8.4 microM. T. b. brucei protein methylase II was sensitive to inhibition by S-adenosyl-L-homocysteine and its analogue sinefungin with apparent Ki values of 12.9 and 1.6 microM, respectively. Using a partially purified preparation, analysis of kinetic data in the presence and absence of sinefungin indicated that this analogue acts as a competitive inhibitor of the S-AdoMet binding site, and as a non-competitive inhibitor of the (protein) histone VIII-S binding site. The possible role of the enzyme in morphological control and its potential as a chemotherapeutic target are discussed. 相似文献
16.
Taylor AB Meyer B Leal BZ Kötter P Schirf V Demeler B Hart PJ Entian KD Wöhnert J 《Nucleic acids research》2008,36(5):1542-1554
Ribosome biogenesis in eukaryotes requires the participation of a large number of ribosome assembly factors. The highly conserved eukaryotic nucleolar protein Nep1 has an essential but unknown function in 18S rRNA processing and ribosome biogenesis. In Saccharomyces cerevisiae the malfunction of a temperature-sensitive Nep1 protein (nep1-1(ts)) was suppressed by the addition of S-adenosylmethionine (SAM). This suggests the participation of Nep1 in a methyltransferase reaction during ribosome biogenesis. In addition, yeast Nep1 binds to a 6-nt RNA-binding motif also found in 18S rRNA and facilitates the incorporation of ribosomal protein Rps19 during the formation of pre-ribosomes. Here, we present the X-ray structure of the Nep1 homolog from the archaebacterium Methanocaldococcus jannaschii in its free form (2.2 A resolution) and bound to the S-adenosylmethionine analog S-adenosylhomocysteine (SAH, 2.15 A resolution) and the antibiotic and general methyltransferase inhibitor sinefungin (2.25 A resolution). The structure reveals a fold which is very similar to the conserved core fold of the SPOUT-class methyltransferases but contains a novel extension of this common core fold. SAH and sinefungin bind to Nep1 at a preformed binding site that is topologically equivalent to the cofactor-binding site in other SPOUT-class methyltransferases. Therefore, our structures together with previous genetic data suggest that Nep1 is a genuine rRNA methyltransferase. 相似文献
17.
P. Blanchard M. S. El Kortbi J. -L. Fourrey F. Lawrence M. Robert-Gero 《Nucleosides, nucleotides & nucleic acids》2013,32(6):1121-1135
Abstract The synthesis of various analogues of sinefungin (l), having structures 2-5, has been developed by means of an original approach which uses radical chemistry. The study of their biological activities revealed that for the antileishmanial effect of sinefungin, the presence of the amino group at C-6′ in the (S)-configuration and the presence of the carboxyl group at C-9′ are necessary. 相似文献
18.
Larbi Dridi Amin Ahmed Ouameur Marc Ouellette 《The Journal of biological chemistry》2010,285(26):19767-19775
S-Adenosylmethionine (AdoMet) is an important methyl group donor that plays a central role in many essential biochemical processes. The parasite Leishmania can both synthesize and transport AdoMet. Leishmania cells resistant to the antifolate methotrexate due to a rearrangement in folate biopterin transporter (FBT) genes were cross-resistant to sinefungin, an AdoMet analogue. FBT gene rearrangements were also observed in Leishmania major cells selected for sinefungin resistance. One of the rearranged FBT genes corresponded to the main AdoMet transporter (AdoMetT1) of Leishmania as determined by gene transfection and gene inactivation experiments. AdoMetT1 was determined to be a high affinity plasma membrane transporter expressed constitutively throughout the growth phases of the parasite. Leishmania cells selected for resistance or naturally insensitive to sinefungin had lower expression of AdoMetT1. A new function in one carbon metabolism, also a pathway of interest for chemotherapeutic interventions, is described for a novel class of membrane proteins found in diverse organisms. 相似文献
19.
ABSTRACT. Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent both in vitro and in vivo. This molecule, structurally related to S-adenosylmethionine, is a good competitive inhibitor of methyltransferases in vitro. The aim of this report is to analyze the impact of sinefungin on methylation pattern and the subcellular localisation of methyl groups and various methylases in promastigotes of Leishmania donovani . We have shown the presence of various methylated macromolecules in different subcellular fractions, with somewhat higher concentration in membrane fraction. In vitro, sinefungin inhibits the three main protein methylases, but in cells cultured in its presence the protein carboxylmethylations are specifically inhibited. 相似文献
20.
While it has been demonstrated that AdoMet is required for DNA cleavage by Type III restriction enzymes, here we show that in the presence of exogenous AdoMet, the head-to-head oriented recognition sites are cleaved only on a supercoiled DNA. On a linear DNA, exogenous AdoMet strongly drives methylation while inhibiting cleavage reaction. Strikingly, AdoMet analogue sinefungin results in cleavage at all recognition sites irrespective of the topology of DNA. The cleavage reaction in the presence of sinefungin is ATP dependent. The site of cleavage is comparable with that in the presence of AdoMet. The use of EcoP15I restriction in presence of sinefungin as an improved tool for serial analysis of gene expression is discussed. 相似文献