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151.
A cofactor-independent phosphoglycerate mutase (PGAM-i) was isolated to homogeneity from monocotyledonous Lilium longiflorum Thunb. Two-dimensional (2D) polyacrylamide gel electrophoresis resolved three PGAM-i forms. This enzyme was originally identified by cross-reactivity to antibodies affinity-purified from 2D gels using human vitronectin (VN). Antibody produced against a denatured protein spot from a 2D gel did not recognize VN protein, but partial protein and DNA sequencing showed similarity of the former protein to maize PGAM-i. Immunoblots from roots, styles, leaves, and anthers showed the presence of PGAM-i in all tissues examined; it was isolated predominantly from the soluble cell fraction, with some present in the insoluble cell fraction. Immunoelectron microscopy demonstrated its localization in the cytoplasm and plastids in root cells near the apical meristem. In addition, immunogold labeling detected signals from the nucleus. The immunohistochemical localization of the enzyme in the nucleus, as well as in the cytosol and plastids, indicates that lily PGAM-i might have multiple functions in the cell.Abbreviations ELISA
enzyme-linked immunosorbent assay
- PGAM
cofactor-dependent phosphoglycerate mutase
- PGAM-i
cofactor independent phosphoglycerate mutase
- TEM
transmission electron microscopy
- 2D
two dimensional
- VN
vitronectin 相似文献
152.
A synthetic metabolic pathway suitable for the production of chorismate derivatives was designed in Escherichia coli. An L-phenylalanine-overproducing E. coli strain was engineered to enhance the availability of phosphoenolpyruvate (PEP), which is a key precursor in the biosynthesis of aromatic compounds in microbes. Two major reactions converting PEP to pyruvate were inactivated. Using this modified E.coli as a base strain, we tested our system by carrying out the production of salicylate, a high-demand aromatic chemical. The titer of salicylate reached 11.5 g/L in batch culture after 48 h cultivation in a 2-liter jar fermentor, and the yield from glucose as the sole carbon source exceeded 40% (mol/mol). In this test case, we found that pyruvate was synthesized primarily via salicylate formation and the reaction converting oxaloacetate to pyruvate. In order to demonstrate the generality of our designed strain, we employed this platform for the production of each of 7 different chorismate derivatives. Each of these industrially important chemicals was successfully produced to levels of 1–3 g/L in test tube-scale culture. 相似文献
153.
The predicted active site of chorismate mutase of baker's yeast Saccharomyces cerevisiae has been studied by continuum electrostatics, molecular surface/volume calculations, and molecular modeling. Our study shows that despite being subject to an allosteric transition, the enzyme's active-site pocket neither decreased in volume nor deformed significantly in shape between the active R state and the inactive T state. We find that the polar atmosphere in the pocket is responsible for the enzyme's affinity. A single amino acid, Glu23, can adequately account for the atmospheric variation. This residue swings into the active-site pocket from the R state to the T state. In the R state, Glu23 on helix H2 doubly pairs with Arg204 and Lys208 of H11, which is packed against H2. In the T state, a slide occurs between H11 and H2 such that Glu23 can no longer interact with Lys208 and competes with Asp24 for interacting with Arg204. Consequently, Glu23 is found in the T state to couple with Arg157, an active-site residue critical to substrate binding. The tandem sliding of H11 in both monomers profoundly changes the interactions in the dimer interface. The loop between H11 and H12 demonstrates the largest conformational change. Hence, we establish a connection between the allosteric transition and the activity of the enzyme. The conformational change in the transition is suggested to propagate into the active-site pocket via a series of polar interactions that result in polarity reversal in the active-site pocket, which regulates the enzyme's activity. Proteins 31:445–452, 1998. © 1998 Wiley-Liss, Inc. 相似文献
154.
为了通过基因工程手段来增加苯丙氨酸的生物产量,利用PCR方法从大肠杆菌中克隆了抗反馈抑制突变型及野生型的pheA基因,进行了核苷酸序列分析,并利用高效的原核表达载体PBV220对pheA基因编码的突变型及野生型分支酸变位酶/预苯酸脱水酶(CM/PD)进行了表达。序列分析表明突变型基因碱基第580位由T变为C,相应氨基酸由Val变为Ala,SDS-PAGE图谱扫描分析表明目的蛋白CM/PD的表达量占全菌体蛋白的43%,占上清总蛋白的57%。酶活性测定表明其CM和 PD活性分别提高了 15.5和6.7倍,产酸量也有了一定的提高,为构建产苯丙氨酸的生物工程菌奠定了基础。 相似文献
155.
Two isoenzymes of chorismate mutase (EC 5.4.99.5) were isolated and partially purified from leaves of diploid (2n=24) Nicotiana silvestris Speg. et Comes and from isogenic cells in a suspension culture originally established from haploid tissue. An isoenzyme denoted CM-1 (M
r=52,000) accounted for the major fraction of total activity recovered from suspension-cultured cells, while isoenzyme CM-2 (M
r=65,000) represented the major fraction of activity recovered from green leaf tissue. The ratio of isoenzyme levels from these two sources differed more than 20-fold. The subcellular location of isoenzyme CM-1 is known to be in the chloroplasts of green leaves or in proplastids of cultured cells, while isoenzyme CM-2 is located in the cytosol. Both isoenzymes were stable during partial purification, possessed broad pH optima for catalysis between 6.0 and 8.0, and were active without denaturation at temperatures at least as high as 45° C. Thiol reagents were unnecessary for either stability or activity of both isoenzymes. The affinity of isoenzyme CM-2 for substrate (K
m=0.24 mM) was almost an order of magnitude better than that of CM-1. The kinetic behavior of isoenzyme CM-1 was influenced by pH, while that of isoenzyme CM-2 was not. At pH 7.2, hyperbolic substrate-saturation curves (K
m=1.7 mM) were obtained for isoenzyme CM-1. At pH 6.1, however, isoenzyme CM-1 displayed relatively weak positive cooperativity, Hill plots yielding an n value of 1.2 At pH 6.1 the half-saturation ([S]0.5) value was 2.5 mM.Abbreviations DEAE
diethylaminoethyl
-
M
r
molecular weight 相似文献
156.
The regulatory properties of chorismate mutase, its cellular localization and isoenzyme pattern were investigated in 23 yeast species. All yeasts contained only a single form of the enzyme, which is localized exclusively in the cytosol. The enzyme activity from all sources was activated 3-(Rhodotorula aurantiaca) to 185-fold (Candida maltosa) by tryptophan. The tryphtophan concentration, which was necessary to obtain half maximum velocity was determined to be between 2 (Pichia guilliermondii) and 95 M (Yarrowia lipolytica). Ten yeast species possessed an enzyme that was inhibited by both phenylalanine and tyrosine. The chorismate mutase from four strains was inhibited only by tyrosine and the enzyme from two species was inhibited by phenylalanine alone. The enzyme inhibition by phenylalanine and tyrosine was completely reversed by tryptophan. Six enzyme sources were not inhibited and theY. lipolytica chorismate mutase was slightly activated by both amino acids. 相似文献
157.
Trypanosoma brucei: activities and subcellular distribution of glycolytic enzymes from differently disrupted cells 总被引:4,自引:0,他引:4
The effect of disruption procedure on the subcellular distribution and the activities of 11 enzymes catalyzing the glycolytic pathway in Trypanosoma brucei has been studied. The activities of the enzymes varied with the lytic procedure used. Maximum specific enzyme activity values were obtained after treatment with saponin whereas digitonin treatment gave the lowest results. The intracellular location of the enzymes was examined by means of differential centrifugation following cell lysis with saponin, Triton X-100, digitonin, or by freezing and thawing. Irrespective of the method of cell lysis employed, the six enzymes, hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, glycerol phosphate dehydrogenase, and glycerokinase, were particulate. Of the remaining 5 enzymes, digitonin liberates only phosphoglycerate mutase (partially); saponin or Triton X-100 liberates phosphoglucose isomerase, phosphoglycerate mutase, enolase, and pyruvate kinase but not glyceraldehyde 3-phosphate dehydrogenase; freezing and thawing acts like saponin or Triton X-100 except that it fails to liberate phosphoglucose isomerase, while cell grinding with silicon carbide liberates only glyceraldehyde phosphate dehydrogenase (partially), phosphoglycerate mutase, enolase, and pyruvate kinase. The relative maximal activities of the enzymes suggest that the rate-limiting steps in glycolysis in T. brucei are the reactions catalyzed by aldolase and phosphoglycerate mutase. 相似文献
158.
Exploring sequence constraints on an interhelical turn using in vivo selection for catalytic activity. 总被引:1,自引:1,他引:0
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G. MacBeath P. Kast D. Hilvert 《Protein science : a publication of the Protein Society》1998,7(2):325-335
The role of interhelical turns in determining protein structure has been investigated previously in relatively simple four-helix-bundle proteins using combinatorial mutagenesis coupled with screening for functional variants. To assess the tolerance to sequence substitution of a short, interhelical turn in a larger, more complicated protein, we have exploited a more sensitive in vivo selection for catalytic activity. Randomization of three solvent-exposed turn residues in Escherichia coli chorismate mutase (Ala65, His66, and His67), followed by selection, indicated that >63% of tripeptides, including some with significantly altered backbone conformations, can functionally replace the native sequence. The increased sensitivity of the catalytic assay allowed optimal sequences to be distinguished from less appropriate ones, revealing a statistically significant preference for hydrophilic residues in solvent-exposed positions. It also enabled investigation of the extent to which either secondary structure or tertiary interactions influence substitution patterns. Randomization of an alpha-helical residue (Lys64), together with the adjacent solvent-exposed tripeptide, Ala65-His66-His67, showed that the secondary structure at position 64 does not limit the range of side chains allowed at this site. In contrast, randomization of a buried turn residue (Leu68), together with the same tripeptide, revealed an extremely strict requirement for hydrophobic aliphatic amino acids at this position. The strong constraint imposed by the tertiary interaction, in contrast to the weak influence of secondary structure, has important implications for protein design. 相似文献
159.
We have isolated the tryptophan auxotrophic mutant strain, PK101, of Pichia guilliermondii. This strain is not defective in any of the tryptophan biosynthetic enzymes, but its chrismate mutase, an enzyme of the phenylalanine-tyrosine biosynthesis, is changed. In comparison with the wild type chorismate mutase, the enzyme of PK101 is characterized by a complete loss of sensitivity to l-phenylalanine inhibition and to a considerable loss of sensitivity to l-tryptophan activation. Furthermore, the chorismate mutase activity of the mutant is more than 7-fold higher in the absence of l-tryptophan than in the wild type. The PK101 enzyme is also changed in the pH optimum and in some kinetic constants. We found an increased intracellular pool of both phenylalanine and tyrosine and a reduced contents of tryptophan in the mutant cells. Our genetic data indicate that the mutant phenotype is dominant over the wild type. 相似文献
160.
The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenylalanine and tyrosine, whereas prephenate dehydratase was very sensitive to inhibition by phenylalanine. Tyrosine was a strong activator of the latter enzyme, whereas anthranilate synthase was inhibited effectively by tryptophan. No clear repression of the synthesis of these enzymes was observed during growth of the organism in the presence of the aromatic amino acids. It is therefore concluded that in Nocardia sp. 239 synthesis of these amino acids is mainly regulated by feedback inhibition. The molecular organization and kinetic properties of DAHP synthase were studied in more detail following its purification. The molecular weight of the native enzyme and its single subunit species were estimated to be 168,000 and 41,000, respectively, suggesting that the enzyme is a tetramer. Apparent K
m values for phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) were 45 and 370 M, respectively. Tryptophan, phenylalanine and tyrosine inhibited DAHP synthase in a competitive manner with respect to E4P, with apparent K
i values of 3, 160 and 180 M, respectively. In addition, tryptophan and E4P (apparent K
i values of 11 and 530 M, respectively) were found to exert an uncompetitive and competitive inhibition, respectively, towards PEP.Abbreviations DAHP
3-deoxy-D-arabino-heptulosonate 7-phosphate
- E4P
erythrose-4-phosphate
- PEP
phosphoenolpyruvate
- RuMP
ribulose monophosphate
- HPLC
high performance liquid chromatography
- FPLC
fast protein liquid chromatography
- SDS
sodium dodecyl sulphate 相似文献