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1.
To gain insight into the evolution of the methionine biosynthesis pathway, in vivo complementation tests were performed. The substrate specificity of three enzymes that intrinsically use different homoserine-esterified substrates and have different sulfur assimilation pathways was examined: two cystathionine gamma-synthases (the Escherichia coli enzyme that naturally utilizes O-succinylhomoserine [OSH]) and the Arabidopsis thaliana enzyme that naturally exploits O-phosphohomoserine [OPH]. Both of these act through the transsulfuration pathway. The third enzyme investigated was O-acetylhomoserine (OAH) sulfhydrylase of Leptospira meyeri, representing the enzyme that utilizes OAH and operates through the direct sulfhydrylation pathway. All the three enzymes were able to utilize OSH and OAH as substrates, with different degrees of efficiency, but only the plant enzyme was able to utilize OPH as a substrate. In addition to their inherent activity in the transsulfuration pathway, the two cystathionine gamma-synthases were also capable of acting in the direct sulfhydrylation pathway. Based on the phylogenic tree and the results of the complementation tests, we suggest that the ancestral gene was able to act as OAH or OSH sulfhydrylase. In some bacteria and plants, this ancient enzyme most probably evolved into a cystathionine gamma-synthase, thereby maintaining the ability to utilize various homoserine-esterified substrates, as well as various sulfur sources, and thus keeping the multisubstrate specificity of its ancestor. In some organisms, this ancestral gene probably underwent a duplication event, which resulted in a cystathionine gamma-synthase and a separate OAH or OSH sulfhydrylase. This led to the development of two parallel pathways of methionine biosynthesis, transsulfuration and direct sulfhydrylation, in these organisms. Although both pathways exist in several organisms, most seem to favor a single specific pathway for methionine biosynthesis in vivo. 相似文献
2.
3.
Seizo Yabuuchi Etsushiro Doi Tadao Hata 《Bioscience, biotechnology, and biochemistry》2013,77(3):687-688
Certain methionine auxotrophs of Arthrobacter paraffineus and Bacillus species produce large amounts of O-acetylhomoserine (OAH). The methionine requirement of these auxotrophs could be satisfied by either cystathionine or homocysteine but not by homoserine. The cell-free extacts from the auxotrophs were found to be deficient in cystathionine ?-synthase activity. OAH and O-succinylhomoserine (OSH) could replace methionine in the auxotrophs which are deficient in homoserine-O-transacetylase. A methionine auxotroph of Corynebacterium glutamicum also produced OAH, and the blocked step in the auxotroph appeared to be between cystathionine and homocysteine.Cell-free extracts of A. paraffineus, C. glutamicum and Bacillus species catalyzed the formation of OAH from acetyl-CoA and homoserine, while a corresponding reaction with succinyl-CoA was not detected. Cystathionine γ-synthases in extracts of C. glutamicum and Bacillus species were specific for OAH, while the enzyme in extract of A. paraffineus was rather specific for OSH though it reacted with OAH to a certain extent.These results indicate that the biosynthesis of l-methionine in these bacteria involves OAH. 相似文献
4.
Cystathionine γ-synthase and β-cystathionase activities were found to be present in cell-free extract of Corynebacterium glutamicum. The reactions catalyzed by cystathionine γ-synthase and β-cystathionase were characterized with respect to Michaelis constant, pH optimum, incubation time and optimal enzyme concentration. Cystathionine γ-synthase was sensitive to the inhibition by S-adenosylmethionine. Formation of cystathionine γ-synthase and β-cystathionase was repressed by the addition of methionine to the growth medium although this repression appeared to be non-coordinate.The regulation of methionine biosynthesis in C. glutamicum was discussed on the basis of these findings. 相似文献
5.
Shuzo Yamagata 《Biochimie》1989,71(11-12)
sulfhydrylase (EC 4.2.99.10) is essential for certain microorganisms, functioning as a homocysteine synthase in the pathway of methionine synthesis. It participates in an alternative pathway of
-homocysteine synthesis for those microbes in which homocysteine is synthesized mainly via cystathionine. The protein can also catalyze the de novo synthesis of
-cysteine and
in some microorganisms. The enzyme possibly recycles the methylthio group of methionine. 相似文献
6.
Aritsune Uchida Taizo Sakata Hajime Kadota 《Bioscience, biotechnology, and biochemistry》2013,77(10):1427-1432
Metabolism of various sulfur compounds in Bacillus subtilis during growth and sporulation was investigated by use of tracer techniques, in an attempt to clarify the mechanism involved in the formation of cystine rich protein of the spore coat.Methionine, homocysteine, cystathionine, cysteine and some inorganic sulfur compounds (sulfate, sulfite and thiosulfate) were utilized by this organism as sulfur sources for its growth and sporulation. Biosynthesis of methionine from sulfate during growth was more or less inhibited by the addition of cysteine, homocysteine or cystathionine to the culture.It is suggested from these results that in Bacillus subtilis methionine is synthesized from sulfate through cysteine, cystathionine and homocysteine as is the case in Salmonella or Neurospora. The results also suggest that the metabolism of sulfur-containing amino acids in Bacillus subtilis is strongly regulated by methionine and homocysteine. 相似文献
7.
In the enteric bacteria Escherichia coli and Salmonella enterica, sulfate is reduced to sulfide and assimilated into the amino acid cysteine; in turn, cysteine provides the sulfur atom for other sulfur-bearing molecules in the cell, including methionine. These organisms cannot use methionine as a sole source of sulfur. Here we report that this constraint is not shared by many other enteric bacteria, which can use either cysteine or methionine as the sole source of sulfur. The enteric bacterium Klebsiella aerogenes appears to use at least two pathways to allow the reduced sulfur of methionine to be recycled into cysteine. In addition, the ability to recycle methionine on solid media, where cys mutants cannot use methionine as a sulfur source, appears to be different from that in liquid media, where they can. One pathway likely uses a cystathionine intermediate to convert homocysteine to cysteine and is induced under conditions of sulfur starvation, which is likely sensed by low levels of the sulfate reduction intermediate adenosine-5'-phosphosulfate. The CysB regulatory proteins appear to control activation of this pathway. A second pathway may use a methanesulfonate intermediate to convert methionine-derived methanethiol to sulfite. While the transsulfurylation pathway may be directed to recovery of methionine, the methanethiol pathway likely represents a general salvage mechanism for recovery of alkane sulfide and alkane sulfonates. Therefore, the relatively distinct biosyntheses of cysteine and methionine in E. coli and Salmonella appear to be more intertwined in Klebsiella. 相似文献
8.
Cysteine and methionine biosynthesis was studied in Pseudomonas putida S-313 and Pseudomonas aeruginosa PAO1. Both these organisms used direct sulfhydrylation of O-succinylhomoserine for the synthesis of methionine but also contained substantial levels of O-acetylserine sulfhydrylase (cysteine synthase) activity. The enzymes of the transsulfuration pathway (cystathionine gamma-synthase and cystathionine beta-lyase) were expressed at low levels in both pseudomonads but were strongly upregulated during growth with cysteine as the sole sulfur source. In P. aeruginosa, the reverse transsulfuration pathway between homocysteine and cysteine, with cystathionine as the intermediate, allows P. aeruginosa to grow rapidly with methionine as the sole sulfur source. P. putida S-313 also grew well with methionine as the sulfur source, but no cystathionine gamma-lyase, the key enzyme of the reverse transsulfuration pathway, was found in this species. In the absence of the reverse transsulfuration pathway, P. putida desulfurized methionine by the conversion of methionine to methanethiol, catalyzed by methionine gamma-lyase, which was upregulated under these conditions. A transposon mutant of P. putida that was defective in the alkanesulfonatase locus (ssuD) was unable to grow with either methanesulfonate or methionine as the sulfur source. We therefore propose that in P. putida methionine is converted to methanethiol and then oxidized to methanesulfonate. The sulfonate is then desulfonated by alkanesulfonatase to release sulfite for reassimilation into cysteine. 相似文献
9.
Stefan Irmler Heike Schäfer Beata Beisert Doris Rauhut & Hélène Berthoud 《FEMS microbiology letters》2009,295(1):67-76
The trans -sulfuration pathways allow the interconversion of cysteine and methionine with the intermediary formation of cystathionine and homocysteine. The genome database of Lactobacillus casei ATCC 334 provides evidence that this species cannot synthesize cysteine from methionine via the trans -sulfuration pathway. However, several L. casei strains use methionine as the sole sulfur source, which implies that these strains can convert methionine to cysteine. Cystathionine synthases and lyases play a crucial role in the trans -sulfuration pathway. By applying proteomic techniques, we have identified a protein in cell-free extracts of L. casei , which showed high homology to a gene product encoded in the genome of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Lactobacillus helveticus but not in the genome of L. casei ATCC 334. The presence of the gene was only found in strains able to grow on methionine as the sole sulfur source. Moreover, two gene variants were identified. Both gene variants were cloned and expressed heterologously in Escherichia coli . The recombinant enzymes exhibited cystathionine lyase activity in vitro and also cleaved cysteine, homocysteine and methionine releasing volatile sulfur compounds. 相似文献
10.
Enzymes implicated in cysteine and methionine metabolism such as cystathionine β‐lyase (CBL; EC 4.4.1.8), a pyridoxal‐5′‐phosphate (PLP)‐dependent carbon–sulfur lyase, have been shown to play a central role in the generation of sulfur compounds. This work describes the unprecedented cloning and characterization of the metC‐cystathionine β‐lyase from the axillary‐isolated strain Staphylococcus haemolyticus AX3, in order to determine its activity and its involvement in amino acid biosynthesis, and in the generation of sulfur compounds in human sweat. The gene contains a cysteine/methionine metabolism enzyme pattern, and also a sequence capable to effect β‐elimination. The recombinant enzyme was shown to cleave cystathionine into homocysteine and to convert methionine into methanethiol at low levels. No odor was generated after incubation of the recombinant enzyme with sterile human axillary secretions; sweat components were found to have an inhibitory effect. These results suggest that the generation of sulfur compounds by Staphylococci and the β‐lyase activity in human sweat are mediated by enzymes other than the metC gene or by the concerted activities of more than one enzyme. 相似文献
11.
Abstract The fission yeast Schizosaccharomyces pombe has a unique organization of sulfur amino acid metabolism: it has two distinct O -acetylhomoserine sulfhydrylases (homocysteine synthases). Similar to Enterobacteriaceae, S. pombe lacks cystathionine β-synthase and cystathionine γ-lyase - the enzymes of the reverse transsulfuration pathway, by which methionine is readily metabolized to cysteine - a likely effector in the sulfur metabolite repression system. Consequently no repression of sulfate assimilation is observed when methionine is added to the growth medium. 相似文献
12.
Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase. 相似文献
13.
In most bacteria, inorganic sulfur is assimilated into cysteine, which provides sulfur for methionine biosynthesis via transsulfurylation. Here, cysteine is transferred to the terminal carbon of homoserine via its sulfhydryl group to form cystathionine, which is cleaved to yield homocysteine. In the enteric bacteria Escherichia coli and Salmonella enterica, these reactions are catalyzed by irreversible cystathionine-gamma-synthase and cystathionine-beta-lyase enzymes. Alternatively, yeast and some bacteria assimilate sulfur into homocysteine, which serves as a sulfhydryl group donor in the synthesis of cysteine by reverse transsulfurylation with a cystathionine-beta-synthase and cystathionine-gamma-lyase. Herein we report that the related enteric bacterium Klebsiella pneumoniae encodes genes for both transsulfurylation pathways; genetic and biochemical analyses show that they are coordinately regulated to prevent futile cycling. Klebsiella uses reverse transsulfurylation to recycle methionine to cysteine during periods of sulfate starvation. This methionine-to-cysteine (mtc) transsulfurylation pathway is activated by cysteine starvation via the CysB protein, by adenosyl-phosphosulfate starvation via the Cbl protein, and by methionine excess via the MetJ protein. While mtc mutants cannot use methionine as a sulfur source on solid medium, they will utilize methionine in liquid medium via a sulfide intermediate, suggesting that an additional nontranssulfurylation methionine-to-cysteine recycling pathway(s) operates under these conditions. 相似文献
14.
Dimethylsulfoniopropionate and Methanethiol Are Important Precursors of Methionine and Protein-Sulfur in Marine Bacterioplankton 总被引:6,自引:4,他引:2
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Ronald P. Kiene Laura J. Linn Jos Gonzlez Mary Ann Moran Jody A. Bruton 《Applied microbiology》1999,65(10):4549-4558
Organic sulfur compounds are present in all aquatic systems, but their use as sources of sulfur for bacteria is generally not considered important because of the high sulfate concentrations in natural waters. This study investigated whether dimethylsulfoniopropionate (DMSP), an algal osmolyte that is abundant and rapidly cycled in seawater, is used as a source of sulfur by bacterioplankton. Natural populations of bacterioplankton from subtropical and temperate marine waters rapidly incorporated 15 to 40% of the sulfur from tracer-level additions of [35S]DMSP into a macromolecule fraction. Tests with proteinase K and chloramphenicol showed that the sulfur from DMSP was incorporated into proteins, and analysis of protein hydrolysis products by high-pressure liquid chromatography showed that methionine was the major labeled amino acid produced from [35S]DMSP. Bacterial strains isolated from coastal seawater and belonging to the α-subdivision of the division Proteobacteria incorporated DMSP sulfur into protein only if they were capable of degrading DMSP to methanethiol (MeSH), whereas MeSH was rapidly incorporated into macromolecules by all tested strains and by natural bacterioplankton. These findings indicate that the demethylation/demethiolation pathway of DMSP degradation is important for sulfur assimilation and that MeSH is a key intermediate in the pathway leading to protein sulfur. Incorporation of sulfur from DMSP and MeSH by natural populations was inhibited by nanomolar levels of other reduced sulfur compounds including sulfide, methionine, homocysteine, cysteine, and cystathionine. In addition, propargylglycine and vinylglycine were potent inhibitors of incorporation of sulfur from DMSP and MeSH, suggesting involvement of the enzyme cystathionine γ-synthetase in sulfur assimilation by natural populations. Experiments with [methyl-3H]MeSH and [35S]MeSH showed that the entire methiol group of MeSH was efficiently incorporated into methionine, a reaction consistent with activity of cystathionine γ-synthetase. Field data from the Gulf of Mexico indicated that natural turnover of DMSP supplied a major fraction of the sulfur required for bacterial growth in surface waters. Our study highlights a remarkable adaptation by marine bacteria: they exploit nanomolar levels of reduced sulfur in apparent preference to sulfate, which is present at 106- to 107-fold higher concentrations. 相似文献
15.
In vivo regulation of de novo methionine biosynthesis in a higher plant (lemna) 总被引:7,自引:7,他引:0
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Administration of methionine to growing Lemna had essentially no effect on accumulation of sulfate sulfur in protein cysteine, but decreased accumulation into cystathionine and its products (homocysteine, methionine, S-methylmethioninesulfonium salt, S-adenosylmethionine, and S-adenosylhomocysteine) to as low as 21% that of control plants, suggesting that methionine regulates its own de novo synthesis at cystathionine synthesis. Methionine caused only a slight reduction (to 80% that of control plants) in the accumulation of sucrose carbon into the 4-carbon moieties of cystathionine and products. This observation was puzzling since cystathionine synthesis proceeds by incorporation of equivalent amounts of sulfur (from cysteine) and 4-carbon moieties (from O-phosphohomoserine). The apparent inconsistency was resolved by the demonstration in Lemna (Giovanelli, Datko, Mudd, Thompson 1983 Plant Physiol 71: 319-326) that de novo synthesis of the methionine 4-carbon moiety occurs not only via the established transsulfuration route from O-phosphohomoserine, but also via the ribose moiety of 5′-methylthioadenosine. It is now clear that the more accurate assessment of the flux of sulfur (and 4-carbon moieties) through transsulfuration is provided by the amount of 35S from 35SO42− that accumulates in cystathionine and its products, rather than by the corresponding measurements with 14C. These studies therefore unequivocally demonstrate in higher plants that methionine does indeed feedback regulate it own de novo synthesis in vivo, and that cystathionine synthesis is a locus for this regulation. 相似文献
16.
Wheeler PR Coldham NG Keating L Gordon SV Wooff EE Parish T Hewinson RG 《The Journal of biological chemistry》2005,280(9):8069-8078
Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine. 相似文献
17.
Kevin Michael Smith Kwang-Myung Cho James C. Liao 《Applied microbiology and biotechnology》2010,87(3):1045-1055
The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways.
Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host’s sensitivity to isobutanol toxicity revealed that
C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols
such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term
batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production
by ∼25% to 4.9 g/L isobutanol in a ∆pyc∆ldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways. 相似文献
18.
A comparison of the rate of ethylene production by apple fruit to the methionine content of the tissue suggests that the sulfur of methionine has to be recycled during its continuous synthesis of ethylene. The metabolism of the sulfur of methionine in apple tissue in relation to ethylene biosynthesis was investigated. The results showed that in the conversion of methionine to ethylene the CH3S-group of methionine is first incorporated as a unit into S-methylcysteine. By demethylation, S-methylcysteine is metabolized to cysteine. Cysteine then donates its sulfur to form methionine, presumably through cystathionine and homocysteine. This view is consistent with the observation that cysteine, homoserine and homocysteine were all converted to methionine, in an order of efficiency from least to greatest. For the conversion to ethylene, methionine was the most efficient precursor, followed by homocysteine and homoserine. Based on these results, a methionine-sulfur cycle in relation to ethylene biosynthesis is presented. 相似文献
19.
【目的】探究丙酮丁醇梭菌半胱氨酸合成代谢途径上铁氧还蛋白和胱硫醚-γ-裂解酶基因的功能。【方法】使用ClosTron系统对半胱氨酸合成途径上的铁氧还蛋白基因(fer)和胱硫醚-γ-裂解酶基因(mccB)进行失活,得到突变株;在不同硫源的培养基中进行分批发酵,分析突变株的生长特点;通过pH控制,使用限磷的连续发酵方法将丙酮丁醇梭菌维持在产酸期和产溶剂期,分析野生型菌株和突变株在连续发酵中的生长情况。【结果】成功构建Δfer和ΔmccB突变株。在分批发酵中,敲除fer基因的突变株无法利用硫酸盐作为硫源,但添加亚硫酸盐或半胱氨酸可以使其恢复生长;在以半胱氨酸为唯一硫源进行分批发酵时,其终浓度1 mmol/L时不会影响野生型与Δfer突变株的生长,但高于1 mmol/L时生长均会受到抑制。在连续发酵中,Δfer突变株不能在产溶剂阶段生长,添加过量的半胱氨酸也不能恢复生长;敲除mccB基因的突变株仍能在添加甲硫氨酸的培养基中生长,但最大OD仅为野生型的57%;相较于野生型,ΔmccB突变株在产酸期和产溶剂期的生长均受到抑制。【结论】fer基因为半胱氨酸合成途径中硫酸盐还原为亚硫酸盐的关键基因,其控制合成的半胱氨酸不能完全由外源的半胱氨酸替代,敲除后对生长的抑制主要表现在连续发酵中的产溶剂阶段。mccB基因参与调控甲硫氨酸转化为半胱氨酸的过程,其敲除会影响甲硫氨酸到半胱氨酸的转化,但不会阻断该生物反应过程。 相似文献
20.
Roles of O-acetyl-L-homoserine sulfhydrylases in micro-organisms 总被引:3,自引:0,他引:3
S Yamagata 《Biochimie》1989,71(11-12):1125-1143
O-Acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) is essential for certain micro-organisms, functioning as a homocysteine synthase in the pathway of methionine synthesis. It participates in an alternative pathway of L-homocysteine synthesis for those microbes in which homocysteine is synthesized mainly via cystathionine. The protein can also catalyze the de novo synthesis of L-cysteine and O-alkyl-L-homoserine in some microorganisms. The enzyme possibly recycles the methylthio group of methionine. 相似文献