共查询到20条相似文献,搜索用时 15 毫秒
1.
Williams SJ Senaratne RH Mougous JD Riley LW Bertozzi CR 《The Journal of biological chemistry》2002,277(36):32606-32615
Bacterial sulfate assimilation pathways provide for activation of inorganic sulfur for the biosynthesis of cysteine and methionine, through either adenosine 5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as intermediates. PAPS is also the substrate for sulfotransferases that produce sulfolipids, putative virulence factors, in Mycobacterium tuberculosis such as SL-1. In this report, genetic complementation using Escherichia coli mutant strains deficient in APS kinase and PAPS reductase was used to define the M. tuberculosis and Mycobacterium smegmatis CysH enzymes as APS reductases. Consequently, the sulfate assimilation pathway of M. tuberculosis proceeds from sulfate through APS, which is acted on by APS reductase in the first committed step toward cysteine and methionine. Thus, M. tuberculosis most likely produces PAPS for the sole use of this organism's sulfotransferases. Deletion of CysH from M. smegmatis afforded a cysteine and methionine auxotroph consistent with a metabolic branch point centered on APS. In addition, we have redefined the substrate specificity of the B. subtilis CysH, formerly designated a PAPS reductase, as an APS reductase, based on its ability to complement a mutant E. coli strain deficient in APS kinase. Together, these studies show that two conserved sequence motifs, CCXXRKXXPL and SXGCXXCT, found in the C termini of all APS reductases, but not in PAPS reductases, may be used to predict the substrate specificity of these enzymes. A functional domain of the M. tuberculosis CysC protein was cloned and expressed in E. coli, confirming the ability of this organism to make PAPS. The expression of recombinant M. tuberculosis APS kinase provides a means for the discovery of inhibitors of this enzyme and thus of the biosynthesis of SL-1. 相似文献
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Kopriva S Fritzemeier K Wiedemann G Reski R 《The Journal of biological chemistry》2007,282(31):22930-22938
Sulfate assimilation provides reduced sulfur for synthesis of the amino acids cysteine and methionine and for a range of other metabolites. Sulfate has to be activated prior to reduction by adenylation to adenosine 5'-phosphosulfate (APS). In plants, algae, and many bacteria, this compound is reduced to sulfite by APS reductase (APR); in fungi and some cyanobacteria and gamma-proteobacteria, a second activation step, phosphorylation to 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is necessary before reduction to sulfite by PAPS reductase (PAPR). We found previously that the moss Physcomitrella patens is unique among these organisms in possessing orthologs of both APR and PAPR genes (Koprivova, A., Meyer, A. J., Schween, G., Herschbach, C., Reski, R., and Kopriva, S. (2002) J. Biol. Chem. 277, 32195-32201). To assess the function of the two enzymes, we compared their biochemical properties by analysis of purified recombinant proteins. APR from Physcomitrella is very similar to the well characterized APRs from seed plants. On the other hand, we found that the putative PAPR preferentially reduces APS. Sequence analysis, analysis of UV-visible spectra, and determination of iron revealed that this new APR, named PpAPR-B, does not contain the FeS cluster, which was previously believed to determine the substrate specificity of the otherwise relatively similar enzymes. The lack of the FeS cluster in PpAPR-B catalysis is connected with a lower turnover rate but higher stability of the protein. These findings show that APS reduction without the FeS cluster is possible and that plant sulfate assimilation is predominantly dependent on reduction of APS. 相似文献
5.
Sulfate assimilation is a pathway providing reduced sulfur for the synthesis of cysteine, methionine, co-enzymes such as iron-sulfur centres, thiamine, lipoic acid, or Coenzyme A, and many secondary metabolites, e.g., glucosinolates or alliins. The pathway is relatively well understood in flowering plants, but very little information exists on sulfate assimilation in basal land plants. Since the finding of a putative 3'-phosphoadenosine 5'-phosphosulfate reductase in PHYSCOMITRELLA PATENS, an enigmatic enzyme thought to exist in fungi and some bacteria only, it has been evident that sulfur metabolism in lower plants may substantially differ from seed plant models. The genomic sequencing of two basal plant species, the Bryophyte PHYSCOMITRELLA PATENS, and the Lycophyte SELAGINELLA MOELLENDORFFII, opens up the possibility to search for differences between lower and higher plants at the genomic level. Here we describe the similarities and differences in the organisation of the sulfate assimilation pathway between basal and advanced land plants derived from genome comparisons of these two species with ARABIDOPSIS THALIANA and ORYZA SATIVA, two seed plants with sequenced genomes. We found differences in the number of genes encoding sulfate transporters, adenosine 5'-phosphosulfate reductase, and sulfite reductase between the lower and higher plants. The consequences for regulation of the pathway and evolution of sulfate assimilation in plants are discussed. 相似文献
6.
Mugford SG Lee BR Koprivova A Matthewman C Kopriva S 《The Plant journal : for cell and molecular biology》2011,65(1):96-105
Sulfur is an essential nutrient for all organisms. Plants take up most sulfur as inorganic sulfate, reduce it and incorporate it into cysteine during primary sulfate assimilation. However, some of the sulfate is partitioned into the secondary metabolism to synthesize a variety of sulfated compounds. The two pathways of sulfate utilization branch after activation of sulfate to adenosine 5'-phosphosulfate (APS). Recently we showed that the enzyme APS kinase limits the availability of activated sulfate for the synthesis of sulfated secondary compounds in Arabidopsis. To further dissect the control of sulfur partitioning between the primary and secondary metabolism, we analysed plants in which activities of enzymes that use APS as a substrate were increased or reduced. Reduction in APS kinase activity led to reduced levels of glucosinolates as a major class of sulfated secondary metabolites and an increased concentration of thiols, products of primary reduction. However, over-expression of this gene does not affect the levels of glucosinolates. Over-expression of APS reductase had no effect on glucosinolate levels but did increase thiol levels, but neither glucosinolate nor thiol levels were affected in mutants lacking the APR2 isoform of this enzyme. Measuring the flux through sulfate assimilation using [(35) S]sulfate confirmed the larger flow of sulfur to primary assimilation when APS kinase activity was reduced. Thus, at least in Arabidopsis, the interplay between APS reductase and APS kinase is important for sulfur partitioning between the primary and secondary metabolism. 相似文献
7.
Synthesis of the sulfur amino acids: cysteine and methionine 总被引:2,自引:0,他引:2
This review will assess new features reported for the molecular and biochemical aspects of cysteine and methionine biosynthesis
in Arabidopsis thaliana with regards to early published data from other taxa including crop plants and bacteria (Escherichia coli as a model). By contrast to bacteria and fungi, plant cells present a complex organization, in which the sulfur network takes
place in multiple sites. Particularly, the impact of sulfur amino-acid biosynthesis compartmentalization will be addressed
in respect to localization of sulfur reduction. To this end, the review will focus on regulation of sulfate reduction by synthesis
of cysteine through the cysteine synthase complex and the synthesis of methionine and its derivatives. Finally, regulatory
aspects of sulfur amino-acid biosynthesis will be explored with regards to interlacing processes such as photosynthesis, carbon
and nitrogen assimilation. 相似文献
8.
Wiedemann G Koprivova A Schneider M Herschbach C Reski R Kopriva S 《Plant molecular biology》2007,65(5):667-676
Sulfate assimilation provides reduced sulfur for the synthesis of the amino acids cysteine and methionine and for a range
of other metabolites. The key step in control of plant sulfate assimilation is the reduction of adenosine 5′-phosphosulfate
to sulfite. The enzyme catalyzing this reaction, adenosine 5′phosphosulfate reductase (APR), is found as an iron sulfur protein
in plants, algae, and many bacteria. In the moss Physcomitrella patens, however, a novel isoform of the enzyme, APR-B, has recently been discovered lacking the co-factor. To assess the function
of the novel APR-B we used homologous recombination to disrupt the corresponding gene in P. patens. The knock-out plants were able to grow on sulfate as a sole sulfur source and the content of low molecular weight thiols
was not different from wild type plants or plants where APR was disrupted. However, when treated with low concentrations of
cadmium the APR-B knockout plants were more sensitive than both wild type and APR knockouts. In wild type P. patens, the two APR isoforms were not affected by treatments that strongly regulate this enzyme in flowering plants. The data thus
suggest that in P. patens APS reduction is not the major control step of sulfate assimilation. 相似文献
9.
Saccharomyces cerevisiae cells grown at 30 degrees C in minimal medium containing methionine lose viability upon transfer to 45 degrees C, whereas cells grown in the absence of methionine survive. Cellular levels of two intermediates in the sulfate assimilation pathway, adenosine 5'-phosphosulfate (APS) and adenosine 5'-phosphosulfate 3'-phosphate, are increased by a posttranslational mechanism after sudden elevation of temperature in yeast cultures grown in the absence of methionine. Yeast cells unable to synthesize APS because of repression by methionine or mutation of the MET3 gene do not survive the temperature shift. Thus, methionine-mediated lethality at elevated temperature is linked to the inability to synthesize APS. The results demonstrate that APS plays an important role in thermotolerance. 相似文献
10.
Sulfonucleotide reductases are a diverse family of enzymes that catalyze the first committed step of reductive sulfur assimilation. In this reaction, activated sulfate in the context of adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is converted to sulfite with reducing equivalents from thioredoxin. The sulfite generated in this reaction is utilized in bacteria and plants for the eventual production of essential biomolecules such as cysteine and coenzyme A. Humans do not possess a homologous metabolic pathway, and thus, these enzymes represent attractive targets for therapeutic intervention. Here we studied the mechanism of sulfonucleotide reduction by APS reductase from the human pathogen Mycobacterium tuberculosis, using a combination of mass spectrometry and biochemical approaches. The results support the hypothesis of a two-step mechanism in which the sulfonucleotide first undergoes rapid nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S-SO(3-)) intermediate. Sulfite is then released in a thioredoxin-dependent manner. Other sulfonucleotide reductases from structurally divergent subclasses appear to use the same mechanism, suggesting that this family of enzymes has evolved from a common ancestor. 相似文献
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Kopriva S Büchert T Fritz G Suter M Benda R Schünemann V Koprivova A Schürmann P Trautwein AX Kroneck PM Brunold C 《The Journal of biological chemistry》2002,277(24):21786-21791
It was generally accepted that plants, algae, and phototrophic bacteria use adenosine 5'-phosphosulfate (APS) for assimilatory sulfate reduction, whereas bacteria and fungi use phosphoadenosine 5'-phosphosulfate (PAPS). The corresponding enzymes, APS and PAPS reductase, share 25-30% identical amino acids. Phylogenetic analysis of APS and PAPS reductase amino acid sequences from different organisms, which were retrieved from the GenBank(TM), revealed two clusters. The first cluster comprised known PAPS reductases from enteric bacteria, cyanobacteria, and yeast. On the other hand, plant APS reductase sequences were clustered together with many bacterial ones, including those from Pseudomonas and Rhizobium. The gene for APS reductase cloned from the APS-reducing cyanobacterium Plectonema also clustered together with the plant sequences, confirming that the two classes of sequences represent PAPS and APS reductases, respectively. Compared with the PAPS reductase, all sequences of the APS reductase cluster contained two additional cysteine pairs homologous to the cysteine residues involved in binding an iron-sulfur cluster in plants. M?ssbauer analysis revealed that the recombinant APS reductase from Pseudomonas aeruginosa contains a [4Fe-4S] cluster with the same characteristics as the plant enzyme. We conclude, therefore, that the presence of an iron-sulfur cluster determines the APS specificity of the sulfate-reducing enzymes and thus separates the APS- and PAPS-dependent assimilatory sulfate reduction pathways. 相似文献
12.
Regulation of sulfate assimilation in Arabidopsis and beyond 总被引:2,自引:0,他引:2
Kopriva S 《Annals of botany》2006,97(4):479-495
BACKGROUND AND AIMS: Sulfate assimilation is a pathway used by prokaryotes, fungi and photosynthetic organisms to convert inorganic sulfate to sulfide, which is further incorporated into carbon skeletons of amino acids to form cysteine or homocysteine. The pathway is highly regulated in a demand-driven manner; however, this regulation is not necessarily identical in various plant species. Therefore, our knowledge of the regulation of sulfate assimilation is reviewed here in detail with emphasis on different plant species. SCOPE: Although demand-driven control plays an essential role in regulation of sulfate assimilation in all plants, the molecular mechanisms of the regulation and the effects of various treatments on the individual enzymes and metabolites are often different. This review summarizes (1) the molecular regulation of sulfate assimilation in Arabidopsis thaliana, especially recent data derived from platform technologies and functional genomics, (2) the co-ordination of sulfate, nitrate and carbon assimilations in Lemna minor, (3) the role of sulfate assimilation and glutathione in plant-Rhizobia symbiosis, (4) the cell-specific distribution of sulfate reduction and glutathione synthesis in C(4) plants, (5) the regulation of glutathione biosynthesis in poplar, (6) the knock-out of the adenosine 5'phosphosulfate reductase gene in Physcomitrella patens and identification of 3'-phosphoadenosyl 5'-phosphosulfate reductase in plants, and (7) the sulfur sensing mechanism in green algae. CONCLUSIONS: As the molecular mechanisms of regulation of the sulfate assimilation pathway are not known, the role of Arabidopsis as a model plant will be further strengthened. However, this review demonstrates that investigations of other plant species will still be necessary to address specific questions of regulation of sulfur nutrition. 相似文献
13.
Cysteine biosynthesis, achieved by the sequential reaction of two enzymes, serine acetyltransferase and O-acetylserine (thiol) lyase (OASTL), represents the final step of sulfur assimilation pathway in plants and bacteria. The two enzymes form a bi-enzymatic cysteine synthase complex through specific protein-protein interactions. To identify the amino acids important for cysteine synthase complex formation, several mutations in bacterial OASTL were designed. Effects of mutagenesis were verified in a yeast two-hybrid model that allowed monitoring both, protein-protein interactions and the enzymatic activity of OASTL. 相似文献
14.
The sulfur assimilation pathway is a key metabolic system in prokaryotes that is required for production of cysteine and cofactors such as coenzyme A. In the first step of the pathway, APS reductase catalyzes the reduction of adenosine 5'-phosphosulfate (APS) to adenosine 5'-phosphate (AMP) and sulfite with reducing equivalents from the protein cofactor, thioredoxin. The primary sequence of APS reductase is distinguished by a conserved iron-sulfur cluster motif, -CC-X( approximately )(80)-CXXC-. Of the sequence motifs that are associated with 4Fe-4S centers, the cysteine dyad is atypical and has generated discussion with respect to coordination as well as the cluster's larger functional significance. Herein, we have used biochemical, spectroscopic, and mass spectrometry analysis to investigate the iron-sulfur cluster and its role in the mechanism of Mycobacterium tuberculosis APS reductase. Site-directed mutagenesis of any cysteine residue within the conserved motif led to a loss of cluster with a concomitant loss in catalytic activity, while secondary structure was preserved. Studies of 4Fe-4S cluster stability and cysteine reactivity in the presence and absence of substrates, and in the free enzyme versus the covalent enzyme-intermediate (E-Cys-S-SO(3)(-)), suggest a structural rearrangement that occurs during the catalytic cycle. Taken together, these results demonstrate that the active site functionally communicates with the iron-sulfur cluster and also suggest a functional significance for the cysteine dyad in promoting site differentiation within the 4Fe-4S cluster. 相似文献
15.
Adenosine 5'-phosphosulfate reductase (APR2) mutation in Arabidopsis implicates glutathione deficiency in selenate toxicity 总被引:1,自引:0,他引:1
Grant K Carey NM Mendoza M Schulze J Pilon M Pilon-Smits EA van Hoewyk D 《The Biochemical journal》2011,438(2):325-335
APR2 is the dominant APR (adenosine 5'-phosphosulfate reductase) in the model plant Arabidopsis thaliana, and converts activated sulfate to sulfite, a key reaction in the sulfate reduction pathway. To determine whether APR2 has a role in selenium tolerance and metabolism, a mutant Arabidopsis line (apr2-1) was studied. apr2-1 plants had decreased selenate tolerance and photosynthetic efficiency. Sulfur metabolism was perturbed in apr2-1 plants grown on selenate, as observed by an increase in total sulfur and sulfate, and a 2-fold decrease in glutathione concentration. The altered sulfur metabolism in apr2-1 grown on selenate did not reflect typical sulfate starvation, as cysteine and methionine levels were increased. Knockout of APR2 also increased the accumulation of total selenium and selenate. However, the accumulation of selenite and selenium incorporation in protein was lower in apr2-1 mutants. Decreased incorporation of selenium in protein is typically associated with increased selenium tolerance in plants. However, because the apr2-1 mutant exhibited decreased tolerance to selenate, we propose that selenium toxicity can also be caused by selenate's disruption of glutathione biosynthesis leading to enhanced levels of damaging ROS (reactive oxygen species). 相似文献
16.
Sulfate uptake in potato tuber discs is inhibited by cysteine and methionine with an 8 h lag period. Cysteine, but not methionine, inhibition can be reversed by washing the treated discs. During the experimental period cysteine is rapidly metabolized, while methionine persists as a free amino acid. Amino acid inhibition of sulfate uptake is overcome by increasing sulfate concentration. The kinetic parameters change suggesting a loss of flexibility of the sulfate uptake system caused by sulfur amino acids. 相似文献
17.
Hanna E MacRae IJ Medina DC Fisher AJ Segel IH 《Archives of biochemistry and biophysics》2002,406(2):275-288
ATP sulfurylase from the hyperthermophilic chemolithotroph Aquifex aeolicus is a bacterial ortholog of the enzyme from filamentous fungi. (The subunit contains an adenosine 5'-phosphosulfate (APS) kinase-like, C-terminal domain.) The enzyme is highly heat stable with a half-life >1h at 90 degrees C. Steady-state kinetics are consistent with a random A-B, ordered P-Q mechanism where A=MgATP, B=SO4(2-), P=PP(i), and Q=APS. The kinetic constants suggest that the enzyme is optimized to act in the direction of ATP+sulfate formation. Chlorate is competitive with sulfate and with APS. In sulfur chemolithotrophs, ATP sulfurylase provides an efficient route for recycling PP(i) produced by biosynthetic reactions. However, the protein possesses low APS kinase activity. Consequently, it may also function to produce PAPS for sulfate ester formation or sulfate assimilation when hydrogen serves as the energy source and a reduced inorganic sulfur source is unavailable. 相似文献
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Rhizobium Meliloti Genes Involved in Sulfate Activation: The Two Copies of Nodpq and a New Locus, Saa 总被引:4,自引:0,他引:4
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The nitrogen-fixing symbiont Rhizobium meliloti establishes nodules on leguminous host plants. Nodulation (nod) genes used for this process are located in a cluster on the pSym-a megaplasmid of R. meliloti. These genes include nodP and nodQ (here termed nodPQ), which encode ATP sulfurylase and APS kinase, enzymes that catalyze the conversion of ATP and SO(4)2- into the activated sulfate form 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in cysteine synthesis. In Rhizobium, PAPS is also a precursor for sulfated and N-acylated oligosaccharide Nod-factor signals that cause symbiotic responses on specific host plants such as alfalfa. We previously found a highly conserved second copy of nodPQ in R. meliloti. We report here the mapping and cloning of this second copy, and its location on the second megaplasmid, pSym-b. The function of nodP2Q2 is equivalent to that of nodP1Q1 in complementation tests of R. meliloti and Escherichia coli mutants in ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase. Mutations in nodP2Q2 do not have as severe an effect on symbiosis or plant host range as do those in nodP1Q1, however, possibly reflecting differences in expression and/or channeling of metabolites to specific enzymes involved in sulfate transfer. Strains mutated or deleted for both copies of nodQ are severely defective in symbiotic phenotypes, but remain prototrophic. This suggests the existence in R. meliloti of a third locus for ATP sulfurylase and APS kinase activities. We have found a new locus saa (sulfur amino acid), which may also encode these activities. 相似文献
19.
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
20.
Kowalska J Osowniak A Zuberek J Jemielity J 《Bioorganic & medicinal chemistry letters》2012,22(11):3661-3664
We describe an efficient and scalable procedure for the chemical synthesis of nucleoside 5'-phosphosulfates (NPS) from nucleoside 5'-phosphorimidazolides and sulfate bis(tributylammonium) salt. Using this method we obtained various NPS with yields ranging from 70-90%, including adenosine 5'-phosphosulfate (APS) and 2',3'-cyclic precursor of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), which are the key intermediates in the assimilation and metabolism of sulfur in all living organisms. 相似文献