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1.
2.
prsB is an allele of the Salmonella typhimurium prsA gene: characterization of a mutant phosphoribosylpyrophosphate synthetase. 总被引:2,自引:1,他引:1
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The Salmonella typhimurium prsB mutation was previously mapped at 45 min on the chromosome, and a prsB strain was reported to produce undetectable levels of phosphoribosylpyrophosphate (PRPP) synthetase activity and very low levels of immunologically cross-reactive protein in vitro (N.K. Pandey and R.L. Switzer, J. Gen. Microbiol, 128:1863-1871, 1982). We have shown by P22-mediated transduction that the prsB gene is actually an allele of prsA, the structural gene for PRPP synthetase, which maps at 35 min. The prsB (renamed prs-100) mutant produces about 20% of the activity and 100% of the cross-reactive material of wild-type strains. prs-100 mutant strains are temperature sensitive, as is the mutant PRPP synthetase in vitro. The prs-100 mutation is a C-to-T transition which results in replacement of Arg-78 in the mature wild-type enzyme by Cys. The mutant PRPP synthetase was purified to greater than 98% purity. It possessed elevated Michaelis constants for both ATP and ribose-5-phosphate, a reduced maximal velocity, and reduced sensitivity to the allosteric inhibitor ADP. The mutant enzyme had altered physical properties and was susceptible to specific cleavage at the Arg-101-to-Ser-102 bond in vivo. It appears that the mutation alters the enzyme's kinetic properties through substantial structural alterations rather than by specific perturbation of substrate binding or catalysis. 相似文献
3.
Chromosomal location of the gene encoding phosphoribosylpyrophosphate synthetase in Escherichia coli 总被引:5,自引:2,他引:3
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B Hove-Jensen 《Journal of bacteriology》1983,154(1):177-184
A mutant of Escherichia coli with a partially defective phosphoribosylpyrophosphate synthetase (ribosephosphate pyrophosphokinase) has been characterized genetically. The genetic lesion causing the altered phosphoribosylpyrophosphate synthetase, prs, was mapped at 26 min on the linkage map by conjugation. Transductional analysis of the prs region established the gene order as purB-fadR-dadR-tre-pth-prs-hemA-trp. Two additional mutations were identified in the mutant: one in gsk, the gene encoding guanosine kinase, and one in lon, conferring a mucoid colony morphology. The contribution of each mutation to the phenotype of the mutant has been evaluated. 相似文献
4.
Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene 总被引:5,自引:0,他引:5
The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting in a DNA fragment of approx. 1.8 kb complementing the E. coli prs mutation. Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an Mr of approx. 40,000. B. subtilis strains harbouring the prs gene in a multicopy plasmid contained up to nine-fold increased PRPP synthetase activity. The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B. subtilis chromosome by homologous recombination. The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA. 相似文献
5.
The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5′ untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains. 相似文献
6.
Mutation in the phosphoribosylpyrophosphate synthetase gene (prs) that results in simultaneous requirements for purine and pyrimidine nucleosides, nicotinamide nucleotide, histidine, and tryptophan in Escherichia coli. 总被引:7,自引:4,他引:3
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B Hove-Jensen 《Journal of bacteriology》1988,170(3):1148-1152
A mutant of Escherichia coli harboring a temperature-labile phosphoribosylpyrophosphate (PRPP) synthetase was characterized. Despite the lack of a detectable PRPP pool or PRPP synthetase activity at 40 degrees C, the strain was fully viable at this temperature as long as guanosine, uridine, histidine, tryptophan, and nicotinamide mononucleotide were all added to the growth medium. Viability of the strain was dependent upon mutations in genes of the nucleoside salvage pathways that improved the utilization of exogenous nucleosides. The properties of the strain are those expected of a PRPP-less strain and suggest that PRPP synthetase is dispensable for E. coli. 相似文献
7.
Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme 总被引:7,自引:0,他引:7
From an Escherichia coli purine auxotroph a mutant defective in phosphoribosylpyrophosphate (PRib-PP) synthetase has been isolated and partially characterized. In contrast to the parental strain, the mutant was able to grow on nucleosides as purine source, whereas growth on purine bases was reduced. Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 muM respectively, compared to 60 muM and 45 muM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib-PP synthetase activity was observed in both strains, although to a lesser extent in the mutant. Our data suggest that the mutant harbors a mutation in the structural gene for PRib-PP synthetase. The mutation responsible for the altered PRib-PP synthetase was located in the purB-hemA region at 26 min on the recalibrated linkage map. 相似文献
8.
An increased activity of phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate is demonstrated in extracts of skin fibroblast cultures derived from a patient with Lesch-Nyhan syndrome. This eccessive response of the phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate results in increased levels of phosphoribosylpyrophosphate and thus contributes to purine overproduction characteristic of this disorder. The level of enzyme response in skin fibroblast extracts from the carrier mother was between activity of the patient and normals, further suggesting the x-linkage of human phosphoribosylpyrophosphate synthetase. 相似文献
9.
A gene (prsA) of Bacillus subtilis involved in a novel, late stage of protein export 总被引:6,自引:0,他引:6
A gene locus of Bacillus subtilis identified by mutations (prs) conferring a defect in protein secretion was cloned from a lambdaGEM-11 expression library. The sites of three closely linked prs mutations (prs-3, prs-29 and prs-40) were found to reside in a 5.3 kb DNA fragment, which also complemented the secretion defect in prs-3 and prs-29 mutants. Partial sequencing of the fragment showed that these three mutations affect one distinct gene (prsA) encoding a putative protein of 292 amino acids (33 kDa). Sequence analysis indicated the PrsA protein to be a lipoprotein located outside the cytoplasmic membrane. Thirty percent identity was shown to the PrtM protein of Lactococcus lactis, which is involved in the maturation of an exported proteinase. The phenotypes of prsA mutants and the structural similarity of PrsA with PrtM suggest that PrsA may have a novel function at a late phase in protein export. 相似文献
10.
目的:基于转酮酶基因缺失菌株MG1655-ΔtktA,研究启动子替换L-组氨酸操纵子前导区及6-磷酸葡萄糖脱氢酶基因zwf、6-磷酸葡萄糖酸脱氢酶基因gnd、PRPP合成酶基因prs的过表达对大肠杆菌产L-组氨酸的影响。方法:通过Red重组系统用T5启动子替换L-组氨酸操纵子前导区;构建gnd和zwf串联表达载体gnd-zwf-pSTV28,prs表达载体prs-pQE30。通过摇瓶发酵,考察上述改造对大肠杆菌积累L-组氨酸的影响。结果:测定结果显示,改造菌株的发酵液中均能实现L-组氨酸积累,平均分别为MG1655-ΔtktA-PT5,60.12 mg/L;MG1655-ΔtktA-PT5(prs-pQE30),66.47mg/L;MG1655-ΔtktA-PT5(zwf-gnd-pSTV28),89.69 mg/L;MG1655-ΔtktA-PT5(prs-pQE30,zwf-gnd-pSTV28),111.56 mg/L。结论:L-组氨酸操纵子前导区的修饰使菌株合成L-组氨酸的能力大大增强,而氧化戊糖磷酸途径的加强和PRPP合成酶活性的提高能够进一步提高产量。 相似文献
11.
The genes encoding both subunits of the succinyl-CoA synthetase of Escherichia coli have been identified as distal genes of the suc operon, which also encodes the dehydrogenase (Elo; sucA) and succinyltransferase (E2o; sucB) components of the 2-oxoglutarate dehydrogenase complex. The newly defined genes express polypeptides of 41 kDa (sucC) and 31 kDa (sucD), corresponding to the beta and alpha subunits of succinyl-CoA synthetase, respectively. The genes are thus located at 16.8 min in the E. coli linkage map, together with the citrate synthase (gltA) and succinate dehydrogenase (sdh) genes, in a cluster of nine citric acid cycle genes: gltA-sdhCDAB-sucABCD. Four deletion strains lacking all of these citric acid cycle enzymes were characterized. The succinyl-CoA synthetase activities of strains harbouring plasmids containing the sucC and sucD genes were amplified some fourfold. Further enzymological studies indicated that expression of succinyl-CoA synthetase is coordinately regulated with 2-oxoglutarate dehydrogenase. 相似文献
12.
Genetic evidence for a repressor of synthesis of cytosine deaminase and purine biosynthesis enzymes in Escherichia coli. 总被引:22,自引:16,他引:6
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Addition of purines to the growth medium of Escherichia coli represses synthesis of cytosine deaminase (codA) and enzymes of purine de novo synthesis. After Tn10 mutagenesis, mutants displaying derepressed levels of cytosine deaminase in the presence of hypoxanthine were isolated. One of these had simultaneously acquired resistance to the hypoxanthine analog 6-mercaptopurine. The mutation purR6::Tn10 was shown to affect de novo synthesis of the purine enzymes glutamine phosphoribosylpyrophosphate amidotransferase (purF) and phosphoribosyl glycinamide synthetase (purD). The mutation was mapped by P1 transduction at 36 min on the E. coli linkage map. A plasmid containing the purR region was obtained by complementation of the purR6::Tn10 mutation. By comparing the restriction maps of the cloned fragment and the E. coli chromosome, the purR gene was found to be located very close to the lpp gene (36.3 min). 相似文献
13.
Ⅰ类内含子(group I intron)是研究RNAs结构与功能关系的理想元件, 在 解释RNA折叠理论、催化机制等方面起着重要作用;对其结构与功能关系的研究也 因此成为一个非常重要的课题. 本研究建立了一个基于卡那霉素抗性进行Ⅰ类内含子结构与功能关系研究系统,将源于海洋蓝细菌Nostoc punctiforme(Npu)核糖核酸还原酶基因(ribonucleotide reductase,Rir)中的1个Ⅰ类内含子插入到pDrive质粒的卡那霉素抗性基因(kanamycin resistance gene,KanR)内构建得pKR12质粒并转化大肠杆菌(E.coli). 只有内含子剪接的阳性克隆才能生成 KanR蛋白并在Kan抗性平板上生长. 结果显示,pKR12转入E.coli后不能在Kan抗性 平板上生长, RT-PCR检测仅可见前体带, 表明插入到KanR中的Npu Rir内含子没有发生剪接. 随后通过易错PCR建立内含子的随机突变库并用Kan抗性筛选进行定向演化, 产生有剪接活性的内含子突变体, RT-PCR检测显示剪接发生. 由于内含子剪接活性的改变可通过Kan抗性变化在LB平板上得以反映, 因此该系统有望成为简单快速地研究Ⅰ类内含子结构与功能关系的有利工具. 相似文献
14.
The soluble acyl-acyl carrier protein synthetase of Vibrio harveyi B392 is a member of the medium chain acyl-CoA synthetase family 总被引:1,自引:0,他引:1
The gene encoding the unique soluble acyl-acyl carrier protein synthetase (AasS) of the bioluminescent Vibrio harveyi strain B392 has been isolated by expression cloning in Escherichia coli.This enzyme catalyzes the ATP-dependent acylation of the thiol of acyl carrier protein (ACP) with fatty acids with chain lengths from C4 to C18. The gene (called aasS) encodes a protein of 60 kDa, a hexahistidine-tagged version of which was readily expressed in E. coli and purified in large quantities. Surprisingly, the sequence of the encoded protein was significantly more similar to that of an acyl-CoA synthetase of the distantly related bacterium, Thermus thermophilus, than to that of the membrane-bound acyl-acyl carrier protein synthetase of E. coli, an enzyme that catalyzes the same reaction from a more closely related organism. Indeed, the AasS sequence can readily be modeled on the known crystal structures of the T. thermophilus acyl-CoA synthetase with remarkably high levels of conservation of the catalytic site residues. To test the possible role of AasS in the fatty aldehyde-dependent bioluminescence pathway of V. harveyi, the chromosomal aasS gene of the organism was disrupted by insertion of a kanamycin cassette by homologous recombination. The resulting aasS::kan strains retained low levels of acyl-acyl carrier protein synthetase consistent with prior indications of a second such activity in this bacterium. The mutant strains grew normally and had normal levels of bioluminescence but were deficient in the incorporation of exogenous octanoic acid into the cellular phospholipids of V. harveyi, particularly at low octanoate concentrations. These data indicate that AasS is responsible for a high-affinity and high-capacity uptake system that efficiently converts exogenous fatty acids into acyl-ACP species competent to enter the fatty acid biosynthetic cycle. 相似文献
15.
Regulation of isoleucine, valine, and leucine biosynthesis and isoleucyl-, valyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined in two mutant strains of Escherichia coli. One mutant was selected for growth resistance to the isoleucine analogue, ketomycin, and the other was selected for growth resistance to both trifluoroleucine and valine. Control of the synthesis of the branched-chain amino acids by repression was altered in both of these mutants. They also exhibited altered control of formation of isoleucyl-tRNA synthetase (EC 6.1.15, isoleucine:sRNA ligase, AMP), valyl-tRNA synthetase (EC 6.1.1.9, valine:sRNA ligase, AMP), and leucyl-tRNA synthetase (EC 6.1.1.4, leucine:sRNA ligase, AMP). These results suggest the existence of a common element for the control of these two classes of enzymes in Escherichia coli. 相似文献
16.
Amino-terminal deletions define a glutamine amide transfer domain in glutamine phosphoribosylpyrophosphate amidotransferase and other PurF-type amidotransferases. 总被引:1,自引:0,他引:1
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A series of deletions was constructed in cloned Escherichia coli purF encoding glutamine phosphoribosylpyrophosphate amidotransferase. These deletions extended into the NH2 terminus of the protein and removed amino acids that are required for glutamine-dependent enzyme activity. Enzyme function, ascribed to the NH3-dependent activity, was retained in deletions that removed up to 237 amino acids. This result supports a model in which PurF-type amidotransferases contain an NH2-terminal glutamine amide transfer domain of approximately 194 to 200 amino acids fused to an aminator domain with NH3-dependent function. 相似文献
17.
Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis. 总被引:5,自引:4,他引:1
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The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E. coli K-12 and in uncapsulated strains such as E. coli O111, was significantly elevated in encapsulated E. coli O10:K5 and O18:K5. This enzyme activity was even higher in an E. coli clone expressing the K5 capsule. This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of the capsular K5 polysaccharide. (i) Expression of the K5 polysaccharide and elevated CMP-KDO synthetase activity were observed with bacteria grown at 37 degrees C but not with cells grown at 20 degrees C or below. (ii) The recovery kinetics of capsule expression of intact bacteria, in vitro K5 polysaccharide-synthesizing activity of bacteria, and CMP-KDO synthetase activity of bacteria after temperature upshift from 18 to 37 degrees C were the same. (iii) Chemicals which inhibit capsule (polysaccharide) expression also inhibited the elevation of CMP-KDO synthetase activity. The chromosomal location of the gene responsible for the elevation of this enzyme activity was narrowed down to the distal segment of the transport region of the K5 expression genes. 相似文献
18.
Mechanisms of accelerated purine nucleotide synthesis in human fibroblasts with superactive phosphoribosylpyrophosphate synthetases 总被引:5,自引:0,他引:5
Concentrations and rates of synthesis of phosphoribosylpyrophosphate (PP-Rib-P) and purine nucleotides were compared in fibroblasts cultured from 5 males with PP-Rib-P synthetase superactivity, 3 normal individuals, and 2 children with severe hypoxanthine-guanine phosphoribosyltransferase deficiency. Although all cell strains with PP-Rib-P synthetase superactivity showed increased PP-Rib-P concentration and generation, increased rates of PP-Rib-P-dependent purine synthetic pathways, and increased purine and pyrimidine nucleoside triphosphate concentrations, two subgroups were discernible. Three fibroblast strains with isolated catalytic defects in PP-Rib-P synthetase showed milder increases in PP-Rib-P concentration (2.5-fold normal) and generation (1.6- to 2.1-fold) and in rates of purine synthesis de novo (1.6- to 2.2-fold) and purine nucleoside triphosphate pools (1.5-fold) than did cells from 2 individuals with combined kinetic defects in PP-Rib-P synthetase, both with purine nucleotide inhibitor-resistance. Values for these processes in the latter two strains were, respectively, 5- to 6-fold, 2.6- to 3.2-fold, 4- to 7-fold, and 1.7- to 2.2-fold those of normal cells. In contrast to cells with catalytic defects, these cells also excreted an abnormally high proportion of labeled purines and resisted purine base-mediated inhibition of PP-Rib-P and purine nucleotide synthesis. Hypoxanthine-guanine phosphoribosyltransferase-deficient cells showed normal regulation of PP-Rib-P synthesis and normal nucleoside triphosphate pools despite increased rates of purine synthesis de novo and of purine excretion. Cells with PP-Rib-P synthetase superactivity thus synthesize purine nucleotides at increased rates as a consequence of increased PP-Rib-P production, despite increased purine nucleotide concentrations. These and additional findings provide evidence that regulation of purine synthesis de novo is effected at both the PP-Rib-P synthetase and amidophosphoribosyltransferase reactions. 相似文献
19.
The four genes required for selenocysteine incorporation were isolated from the gram-positive, amino acid-fermenting anaerobe Eubacterium acidaminophilum, which expresses various selenoproteins of different functions. The sel genes were located in an unique organization on a continuous fragment of genomic DNA in the order selD1 (selenophosphate synthetase 1), selA (selenocysteine synthase), selB (selenocysteine-specific elongation factor), and selC (selenocysteine-specific tRNA). A second gene copy, encoding selenophosphate synthetase 2 (selD2), was present on a separate fragment of genomic DNA. SelD1 and SelD2 were only 62.9% identical, but the two encoding genes, selD1 and selD2, contained an in-frame UGA codon encoding selenocysteine, which corresponds to Cys-17 of Escherichia coli SelD. The function of selA, selB, and selC from E. acidaminophilum was investigated by complementation of the respective E. coli deletion mutant strains and determined as the benzyl viologen-dependent formate dehydrogenase activity in these strains after anaerobic growth in the presence of formate. selA and selC from E. acidaminophilum were functional and complemented the respective mutant strains to 83% (selA) and 57% (selC) compared to a wild-type strain harboring the same plasmid. Complementation of the E. coli selB mutant was only observed when both selB and selC from E. acidaminophilum were present. Under these conditions, the specific activity of formate dehydrogenase was 55% of that of the wild type. Transformation of this selB mutant with selB alone was not sufficient to restore formate dehydrogenase activity. 相似文献
20.
Several regulators of methionine biosynthesis have been reported in Escherichia coli, which might represent barriers to the production of excess l-methionine (Met). In order to examine the effects of these factors on Met biosynthesis and metabolism, deletion mutations of the methionine repressor (metJ) and threonine biosynthetic (thrBC) genes were introduced into the W3110 wild-type strain of E. coli. Mutations of the metK gene encoding S-adenosylmethionine synthetase, which is involved in Met metabolism, were detected in 12 norleucine-resistant mutants. Three of the mutations in the metK structural gene were then introduced into metJ and thrBC double-mutant strains; one of the resultant strains was found to accumulate 0.13 g/liter Met. Mutations of the metA gene encoding homoserine succinyltransferase were detected in alpha-methylmethionine-resistant mutants, and these mutations were found to encode feedback-resistant enzymes in a 14C-labeled homoserine assay. Three metA mutations were introduced, using expression plasmids, into an E. coli strain that was shown to accumulate 0.24 g/liter Met. Combining mutations that affect the deregulation of Met biosynthesis and metabolism is therefore an effective approach for the production of Met-excreting strains. 相似文献