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1.
Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli 总被引:1,自引:0,他引:1
L J Kimlova C Pyne K Keshavjee J Huy G Beebakhee A L Bognar 《Archives of biochemistry and biophysics》1991,284(1):9-16
The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme. 相似文献
2.
Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.
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The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products. 相似文献
3.
Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments. The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme. In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide. The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP. A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol. A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype. A met7 shm2 strain is nonviable. In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam). All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain. The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine. Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP. Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene. 相似文献
4.
Altered regulation of the glnRA operon in a Bacillus subtilis mutant that produces methionine sulfoximine-tolerant glutamine synthetase. 总被引:1,自引:1,他引:0
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A Bacillus subtilis mutant that produced glutamine synthetase (GS) with altered sensitivity to DL-methionine sulfoximine was isolated. The mutation, designated glnA33, was due to a T.A-to-C.G transition, changing valine to alanine at codon 190 within the active-site C domain. Altered regulation was observed for GS activity and antigen and mRNA levels in a B. subtilis glnA33 strain. The mutant enzyme was 28-fold less sensitive to DL-methionine sulfoximine and had a 13.0-fold-higher Km for hydroxylamine and a 4.8-fold-higher Km for glutamate than wild-type GS did. 相似文献
5.
Threonyl-transfer ribonucleic acid synthetase and the regulation of the threonine operon in Escherichia coli.
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Two threonine-requiring mutants with derepressed expression of the threonine operon were isolated from an Escherichia coli K-12 strain containing two copies of the thr operon. One of them carries a leaky mutation in ilvA (the structural gene for threonine deaminase), which creates an isoleucine limitation and therefore derepression of the thr operon. In the second mutant, the enzymes of the thr operon were not repressed by threonine plus isoleucine; the threonyl-transfer ribonucleic acid(tRNA) synthetase from this mutant shows an apparent Km for threonine 200-fold higher than that of the parental strain. The gene, called thrS, coding for threonyl-tRNA synthetase was located around 30 min on the E. coli map. The regulatory properties of this mutant imply the involvement of charged threonyl-tRNA or threonyl-tRNA synthetase in the regulation of the thr operon. 相似文献
6.
Suda N Nakagawa Y Kikuchi A Sawada M Takami Y Funahashi HO Nakao A Yoshida S Suzuki M 《Biochemical and biophysical research communications》2003,303(1):46-51
We studied the mutation effect of one of the putative loop residues Thr792 in human DNA topoisomerase II alpha (TOP2 alpha). Thr792 mutants were expressed from high or low copy plasmids in a temperature sensitive yeast strain deficient in TOP2 (top2-1). When expressed from a high copy plasmid, mutants with small side chains complemented the yeast defect; however, from a low copy plasmid, only wild-type, Ser, and Cys substitution mutants complemented the yeast defect. Interestingly, at the permissive temperature other mutants (e.g., Val, Gly, and Glu substitutions) showed the dominant negative effect to the top2-1 allele, which was not observed by the control alpha 4-helix mutants. T792E mutant was 10-fold less active than wild-type and the T792P had no decatenation activity in vitro. These results suggest that Thr792 in human TOP2 alpha is involved in enzyme catalysis. 相似文献
7.
Bacillus subtilis glutamine synthetase mutants pleiotropically altered in glucose catabolite repression. 总被引:24,自引:20,他引:4
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Strain SF22, a glutamine-requiring (Gln-) mutant of Bacillus subtilis SMY, is likely to have a mutation in the structural gene for glutamine synthetase, since this strain synthesized 22 to 55% as much glutamine synthetase antigen as did wild-type cells in a 10-min period but had less than 3% of wild-type glutamine synthetase enzymatic activity. The expression of several genes subject to glucose catabolite repression was altered in the Gln- mutant. The induced levels of alpha-glucosidase, histidase, and aconitase were 3.5- to 4-fold higher in SF22 cells than in wild-type cells grown in glucose-glutamine medium, and citrate synthase levels were 8-fold higher in the Gln- mutant than in wild-type cells. The relief of glucose catabolite repression in the Gln- mutant may result from poor utilization of glucose. Examination of the intracellular metabolite pools of cells grown in glucose-glutamine medium showed that the glucose-6-phosphate pool was 2.5-fold lower, the pyruvate pool was 4-fold lower, and the 2-ketoglutarate pool was 2.5-fold lower in the Gln- cells than they were in wild-type cells. Intracellular levels of glutamine were sixfold higher in the Gln- mutant than in wild-type cells. Measurements of enzymes involved in glutamine transport and utilization showed that the elevated pools of glutamine in the Gln- mutant resulted from a threefold increase in glutamine permease and a fivefold decrease in glutamate synthase. The pleiotropic effect of the gln-22 mutation on the expression of several genes suggests that either the glutamine synthetase protein or its enzymatic product, glutamine, is involved in the regulation of several metabolic pathways in B. subtilis. 相似文献
8.
Mutants affecting tRNA(Phe) from Escherichia coli. Studies of the suppression of thermosensitive phenylalanyl-tRNA synthetase 总被引:2,自引:0,他引:2
Four mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild-type pheV to complement the thermosensitive defect in a mutant of phenylalanyl-tRNA synthetase. One of these mutants, leading to the change C2----U2 in tRNA(Phe), is expressed about 10-fold lower in transformed cells than wild-type pheV. This mutant, unlike the remaining three (G15----A15, G44----A44, m7G46----A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number. The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity. A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl-tRNA synthetase. The mutant gene pheV (U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second-site mutation outside the tRNA structural gene, in the discriminator region. This mutation, C(-6)----T(-6), restores expression of the mutant U2 to about the level of wild-type tRNA(Phe). 相似文献
9.
L-Methionine SR-sulfoximine-resistant glutamine synthetase from mutants of Salmonella typhimurium 总被引:3,自引:0,他引:3
Two mutants of Salmonella typhimurium resistant to growth inhibition by the glutamine synthetase transition state analog, L-methionine SR-sulfoximine, were isolated and characterized. These mutants are glutamine bradytrophs and cannot use growth rate-limiting nitrogen sources. Although this phenotype resembles that of mutants with lesions in the regulatory gene for glutamine synthetase, glnG, these mutations do not lie in the glnG gene. Purification and characterization of the glutamine synthetase from one of the mutants and a control strain demonstrated that the mutant enzyme is defective in the reverse gamma-glutamyltransferase activity but has biosynthetic activity that is resistant to inhibition by L-methionine SR-sulfoximine. The mutant enzyme also has a 4.4-fold higher apparent Km for glutamate (0.2 mM versus 2.1 mM, respectively) and a 13.8-fold higher Km for NH3 (6.4 mM versus 0.46 mM) than the enzyme from the control. These data show that the glutamine synthetase protein has been altered by this mutation, designated as glnA982, and suggest that the L-methionine SR-sulfoximine resistance is conferred by a change in the NH3 binding domain of the enzyme. 相似文献
10.
An isoleucine auxotroph of Salmonella typhimurium was derived from a merodiploid strain (containing the F-14 episome from Escherichia coli) that contained two copies of the structural genes concerned with isoleucine and valine biosynthesis. A haploid derivative, strain TU6001, having the same growth properties as the original merodiploid mutant was found to have normal biosynthetic enzymes and an altered isoleucyl-tRNA synthetase. The K
m for isoleucine was increased by about 200-fold over that for the wild-type enzyme. All five enzymes in the isoleucine and valine biosynthetic pathway were derepressed relative to wild-type enzyme levels. A partial revertant of strain TU6001 was isolated which had properties that were intermediate between those of the mutant and the wild type (i.e., intermediate growth dependence on exogenous isoleucine, intermediate activity of isoleucyl-tRNA synthetase, and intermediate derepression of biosynthetic enzymes). The properties of strain TU6001 were demonstrated to be simultaneously transferable by transduction (using PLT22 H4 bacteriophage) of a single genetic locus, linked to pyr A, which has been designated ilv S. It is concluded that some function of the isoleucyl-tRNA synthetase is important in repression of the isoleucine and valine biosynthetic enzymes.Supported by grant GM 12522 from the National Institute of General Medical Sciences, U.S. Public Health Service. J. M. B. received a U.S. Public Health Service Postdoctoral Fellowship 1-F02-GM-30, 650-02. 相似文献
11.
Stephen Y K Seah K Linda Britton David W Rice Yasuhisa Asano Paul C Engel 《European journal of biochemistry》2003,270(23):4628-4634
Through comparison with the high-resolution structure of Clostridium symbiosum glutamate dehydrogenase, the different substrate specificities of the homologous enzymes phenylalanine dehydrogenase and leucine dehydrogenase were attributed to two residues, glycine 124 and leucine 307, in Bacillus sphaericus phenylalanine dehydrogenase, which are replaced with alanine and valine in leucine dehydrogenases. As predicted, making these substitutions in phenylalanine dehydrogenase decreased the specific activity towards aromatic substrates and enhanced the activity towards some aliphatic amino acids in standard assays with fixed concentrations of both substrates. This study did not, however, distinguish effects on affinity from those on maximum catalytic rate. A fuller kinetic characterization of the single- and double-mutant enzymes now reveals that the extent of the shift in specificity was underestimated in the earlier study. The maximum catalytic rates for aromatic substrates are reduced for all the mutants, but, in addition, the apparent Km values are higher for the single-mutant G124A and double-mutant G124A/L307V compared with the wild-type enzyme. Conversely, specificity constants (kcat/Km) for the nonpolar aliphatic amino acids and the corresponding 2-oxoacids for the mutants are all markedly higher than for the wild type, with up to a 40-fold increase for l-norvaline and a 100-fold increase for its 2-oxoacid in the double mutant. In some cases a favourable change in Km was found to outweigh a smaller negative change in kcat. These results emphasize the risk of misjudging the outcome of protein engineering experiments through too superficial an analysis. Overall, however, the success of the predictions from molecular modelling indicates the usefulness of this strategy for engineering new specificities, even in advance of more detailed 3D structural information. 相似文献
12.
Escherichia coli K-12 Mutants Altered in the Transport of Branched-Chain Amino Acids 总被引:4,自引:6,他引:4
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Two mutants of Escherichia coli K-12 are described which are resistant to the inhibition that valine exerts on the growth of E. coli. These mutants have lesions at two different loci on the chromosome. One of them, brnP, is linked to leu (87% cotransduction) and is located between leu and azi represented on the map at 1 min; the other, brnQ, is linked to phoA (96% cotransduction), probably between proC and phoA and represented at 10 min. These mutants are resistant to valine inhibition but are sensitive to dipeptides containing valine. Since it is known that dipeptides are taken up by E. coli through a transport system(s) different from those used by amino acids, this sensitivity to the peptides suggests an alteration in the active transport of valine. The mutants are resistant to valine only if leucine is present in the growth medium; the uptake of valine is less in both mutants than it is in wild-type E. coli, and it is reduced even further if leucine is present. Under these conditions the total uptake of valine is almost completely abolished in the brnQ mutant. The brnP mutant takes up about 60% as much valine as does the wild type, but no exogenous valine is incorporated into proteins. The apparent K(m) and V(max) of isoleucine, leucine, and valine for the transport system are reported; the brnP mutant, when compared to the wild type, has a sevenfold higher K(m) for isoleucine and a 17-fold lower K(m) for leucine; the V(max) for the three amino acids is reduced in the brnQ mutant, up to 20-fold for valine. The transport of arginine, aspartic acid, glycine, histidine, and threonine is not altered in the brnQ mutant under conditions in which that of the branched amino acids is. Evidence is reported that O-methyl-threonine enters E. coli through the transport system for branched amino acids, and that thiaisoleucine does not. 相似文献
13.
Mutants of Salmonella typhimurium defective in transport of branched-chain amino acids 总被引:2,自引:1,他引:1
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K Kiritani 《Journal of bacteriology》1974,120(3):1093-1101
Five mutants, CD15, CD31, CE4, CE5, and CE14, defective in the transport of branched-chain amino acids were isolated as glycyl-l-isoleucine and glycyl-l-valine requirers from an isoleucine-valine-requiring mutant, KA931, of Salmonella typhimurium LT2. Although these transport mutants do not grow in minimal medium supplemented with isoleucine (10 mug/ml) and valine (20 mug/ml), where the parent strain, KA931, grows normally, they do when the supplements are increased 10-fold in concentration, and the growth rate becomes comparable to that of the parent strain. When the apparent K(m) and V(max) for uptake of isoleucine, valine, and leucine in the transport mutants were compared to those of KA931, the K(m) was generally lower in the mutants, and the V(max) for isoleucine and leucine decreased to one-fourth to one-seventh and for valine one-eighth to one-fifteenth of that in the parent. In all mutants except CE5, the transport of methionine, arginine, threonine, and glycine was normal. The transport of threonine in CE5 appeared to be slightly impaired. In addition to the original mutation in the ilvC locus, the transport mutant has a mutation at the ilvT locus, which is closely linked to proC and may be located on the side of proC proximal to purE. About 26-fold difference in values of the co-transduction is noted in reciprocal transductions between KA502 and CD15 strains. 相似文献
14.
Isoleucine auxotrophy as a consequence of a mutationally altered isoleucyl-transfer ribonucleic acid synthetase 总被引:19,自引:10,他引:9
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Among mutants which require isoleucine, but not valine, for growth, we have found two distinguishable classes. One is defective in the biosynthetic enzyme threonine deaminase (l-threonine hydro-lyase, deaminating, EC 4.2.1.16) and the other has an altered isoleucyl transfer ribonucleic acid (tRNA) synthetase [l-isoleucine: soluble RNA ligase (adenosine monophosphate), EC 6.1.1.5]. The mutation which affects ileS, the structural gene for isoleucyl-tRNA synthetase, is located between thr and pyrA at 0 min on the map of the Escherichia coli chromosome. This mutationally altered isoleucyl-tRNA synthetase has an apparent K(m) for isoleucine ( approximately 1 mm) 300-fold higher than that of the enzyme from wild type; on the other hand, the apparent V(max) is altered only slightly. When the mutationally altered ileS allele was introduced into a strain which overproduces isoleucine, the resulting strain could grow without addition of isoleucine. We conclude that the normal intracellular isoleucine level is not high enough to allow efficient charging to tRNA(Ile) by the mutant enzyme because of the K(m) defect. A consequence of the alteration in isoleucyl-tRNA synthetase was a fourfold derepression of the enzymes responsible for isoleucine biosynthesis. Thus, a functional isoleucyl-tRNA synthetase is needed for isoleucine to act as a regulator of its own biosynthesis. 相似文献
15.
A L Bognar C Osborne B Shane S C Singer R Ferone 《The Journal of biological chemistry》1985,260(9):5625-5630
The Escherichia coli gene for folylpolyglutamate synthetase-dihydrofolate synthetase was localized to plasmids pLC22-45, 24-31, and 28-44 of the Clarke-Carbon E. coli colony bank (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) by screening the bank by replica mating with an E. coli folC mutant. The folC gene was subcloned from pLC22-45 and inserted into a high copy number plasmid containing the lambda replication control region under the control of the temperature-sensitive cI857 repressor and into a high expression plasmid containing the lambda PL promoter and the cI857 repressor. The folC structural gene was located on a 1.52-kilobase PvuI fragment, sufficient to code for a protein of maximum Mr 55,000. E. coli transformants containing the recombinant plasmids, when induced by culturing at 42 degrees C, had folylpolyglutamate synthetase and dihydrofolate synthetase levels that were 100- to 400-fold higher than in wild type strains and which represented up to 4% of the soluble cell protein. The E. coli folylpolyglutamate synthetase-dihydrofolate synthetase has been purified to homogeneity from the transformants. Both activities are catalyzed by a single protein of Mr 47,000. Some kinetic properties of the enzymes and a new spectrophotometric method for assaying dihydrofolate synthetase activity are described. 相似文献
16.
Robert Twarog 《Journal of bacteriology》1972,111(1):37-46
Regulation of four of the enzymes required for isoleucine and valine biosynthesis in Acinetobacter was studied. A three- to fourfold derepression of acetohydroxyacid synthetase was routinely observed in two different wild-type strains when grown in minimal medium relative to cells grown in minimal medium supplemented with leucine, valine, and isoleucine. A similar degree of synthetase derepression was observed in appropriately grown isoleucine or leucine auxotrophs. No significant derepression of threonine deaminase or transaminase B occurred in either wild-type or mutant cells grown under a variety of conditions. Three amino acid analogues were tested with wild-type cells; except for a two- to threefold derepression of dihydroxyacid dehydrase when high concentrations of aminobutyric acid were added to the medium, essentially the same results were obtained. Experiments showed that threonine deaminase is subject to feedback inhibition by isoleucine and that valine reverses this inhibition. Cooperative effects in threonine deaminase were demonstrated with crude extracts. The data indicate that the synthesis of isoleucine and valine in Acinetobacter is regulated by repression control of acetohydroxyacid synthetase and feedback inhibition of threonine deaminase and acetohydroxyacid synthetase. 相似文献
17.
Spontaneous and ICR191-A-induced Frameshift Mutations in the A Gene of Escherichia coli Tryptophan Synthetase 总被引:10,自引:6,他引:4
Thiaisoleucine-resistant mutants of Escherichia coli strain K-12 which exhibited reduced isoleucyl soluble ribonucleic acid synthetase activity were isolated. Resistance was apparently achieved by the selection of a synthetase with a 10-fold decrease in apparent affinity for thiaisoleucine. This mutation also resulted in a 2.5-fold decrease in apparent affinity for the natural substrate, l-isoleucine, and less activity than found in wild type. The mutants grew more slowly than wild type and were derepressed for three of the five enzymes in the pathways to isoleucine and valine. 相似文献
18.
An analysis of the side chain requirement at position 177 within the lactose permease which confers the ability to recognize maltose. 总被引:1,自引:0,他引:1
In previous work (Brooker, R. J., and Wilson, T. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3959-3963), lactose permease mutants were isolated which possessed an enhanced recognition for maltose. In some of these mutants, the wild-type alanine residue at position 177 was changed to valine or threonine. To gain further insight into the side chain requirement at position 177 that confers maltose recognition, further substitutions of isoleucine, leucine, phenylalanine, proline, and serine have been made via site-directed mutagenesis. Permeases containing alanine or serine exhibited poor maltose recognition whereas those containing isoleucine, leucine, phenylalanine, proline, or valine showed moderate or good recognition. As far as galactosides are concerned, the Val-177, Pro-177, and Ser-177 mutants were able to transport lactose as well as, or slightly better than, the wild-type strain. The other mutants displayed moderately reduced levels of lactose transport. For example, the Phe-177 mutant, which was the most defective, showed a level of downhill transport which was approximately 20% that of the wild-type strain. In uphill transport assays, all of the position 177 mutants were markedly defective in their ability to accumulate beta-D-thiomethylgalactopyranoside against a concentration gradient. Finally, the position 177 mutants were analyzed for their ability to catalyze an H+ leak. Interestingly, even though the wild-type permease does not leak H+ across the bacterial membrane, all of the position 177 mutants were shown to transport H+ in the absence of sugars. For most of the mutants, this H+ leak was blocked by the addition of beta-D-thiodigalactoside. Overall, these results are discussed with regard to the effects of position 177 substitutions on the sugar recognition site and H+ transport. 相似文献
19.
Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942. 总被引:23,自引:14,他引:9
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Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp. 相似文献
20.
The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed. 相似文献