共查询到20条相似文献,搜索用时 15 毫秒
1.
Mino K Hiraoka K Imamura K Sakiyama T Eisaki N Matsuyama A Nakanishi K 《Bioscience, biotechnology, and biochemistry》2000,64(9):1874-1880
Some properties of serine acetyltransferases (SATs) from Escherichia coli, deleting 10-25 amino acid residues from the C-terminus (SATdeltaC10-deltaC25) were investigated. The specific activity depended only slightly on the length of the C-terminal region deleted. Although the sensitivity of SATdeltaC10 to inhibition by L-cysteine was similar to that for the wild-type SAT, it became less with further increases in the length of the amino acid residues deleted. SATdeltaC10 was inactivated on cooling to 0 degrees C and dissociated into dimers or trimers in the same manner as the wild-type SAT, but Met-256-le mutant SAT as well as SATdeltaC14, SATdeltaC20, and SATdeltaC25 were stable. Since SATdeltaC10, SATdeltaC14, and SATdeltaC25 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A) in a way similar to SATdeltaC20, it was indicated that 10 amino acid residues or fewer from the C-terminus of the wild-type SAT are responsible for the complex formation with OASS-A. The C-terminal peptide of the 10 amino acid residues interacted competitively with OASS-A with respect to OAS although its affinity was much lower than that for the wild-type SAT. 相似文献
2.
Mino K Yamanoue T Sakiyama T Eisaki N Matsuyama A Nakanishi K 《Bioscience, biotechnology, and biochemistry》1999,63(1):168-179
Incubation of serine acetyltransferase (SAT) from Escherichia coli at 25 degrees C in the absence of protease inhibitors yielded a truncated SAT. The truncated SAT was much less sensitive to feedback inhibition than the wild-type SAT. Analyses of the N- and C-terminal amino acid sequences found that the truncated SAT designated as SAT delta C20 was a resultant form of the wild-type SAT cleaved between Ser 253 and Met 254, deleting 20 amino acid residues from the C-terminus. Based on these findings, we constructed a plasmid containing an altered cysE gene encoding the truncated SAT. SAT delta C20 was produced using the cells of E. coli JM70 transformed with the plasmid and purified to be homogeneous on an SDS-polyacrylamide gel. Properties of the purified SAT delta C20 were investigated in comparison with those of the wild-type SAT and Met-256-Ile mutant SAT, which was isolated by Denk and B?ck but not purified (J. Gen. Microbiol., 133, 515-525 (1987)). SAT delta C20 was composed of four identical subunits like the wild-type SAT and Met-256-Ile mutant SAT. Specific activity, optimum pH for reaction, thermal stability, and stability to reagents for SAT delta C20 were similar those for the wild-type SAT and Met-256-Ile mutant SAT. However, SAT delta C20 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A), a counterpart of the cysteine synthetase and did not reduce OASS activity in contrast to the wild-type SAT and Met-256-Ile mutant SAT. 相似文献
3.
T Nozaki T Asai L B Sanchez S Kobayashi M Nakazawa T Takeuchi 《The Journal of biological chemistry》1999,274(45):32445-32452
The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites. 相似文献
4.
L-Cysteine is an important amino acid in terms of its industrial applications. The biosynthesis of L-cysteine in enteric bacteria is regulated through the feedback inhibition by L-cysteine of L-serine O-acetyltransferase (SAT), a key enzyme in L-cysteine biosynthesis. We recently found that L-cysteine is overproduced in Escherichia coli strains expressing a gene encoding feedback inhibition-insensitive SAT. Further improvements in L-cysteine production are expected by the use of SAT with high stability. We report here the sat1 gene encoding SAT of an extreme thermophile, Thermus thermophilus HB8. The sat1 gene was cloned and overexpressed in E. coli cells based on the genome sequence in T. thermophilus HB8. The predicted amino acid sequence consists of 295 amino acids and is homologous to other O-acetyltransferase members. In particular, the carboxyl-terminal region shares approximately 30% identities with SATs found in bacteria and plants, despite showing only about 15% identity in the overall sequence. Enzymatic analysis and an atomic absorption study of the purified recombinant proteins revealed that the enzyme is highly activated by Co(2+) or Ni(2+), and contains Zn(2+) and Fe(2+). These results indicate that the T. thermophilus SAT is a novel type of enzyme different from other members of this protein family. 相似文献
5.
Zhao C Moriga Y Feng B Kumada Y Imanaka H Imamura K Nakanishi K 《Biochemical and biophysical research communications》2006,341(4):911-916
Cysteine synthase from Escherichia coli is a bienzyme complex comprised of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase A. The site of interaction of a SAT molecule was investigated by gel chromatography and surface plasmon technique using various mutant-type SATs, to better understand the mechanism involved in complex formation. The C-terminus of SAT, Ile 273, along with Glu 268 and Asp 271, was found to be essential for complex formation. The effects of O-acetyl-L-serine and sulfide on the affinity for the complex formation were also studied using a surface plasmon technique. 相似文献
6.
Zhao C Kumada Y Imanaka H Imamura K Nakanishi K 《Protein expression and purification》2006,47(2):607-613
O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E. coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-B was characterized and its properties compared with OASS-A. OASS-B did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis. 相似文献
7.
Serine acetyltransferase (SAT) from Escherichia coli is subject to feedback inhibition by L-cysteine. A mutant was isolated which excretes L-cysteine because of a lesion in cysE, the structural gene for SAT, rendering the enzyme less feedback sensitive. To analyse the structural basis for this mutation the cysE genes both from wild-type E. coli and the mutant strain were cloned and their nucleotide sequences determined. The cysE gene contained an open reading frame consisting of 819 bp, equivalent to a protein of 273 amino acids. The mutant gene showed a single base change in position 767 resulting in a methionine to isoleucine substitution. A causal connection between this SAT sequence alteration, feedback insensitivity and L-cysteine excretion was demonstrated. The SAT from the wild-type strain was purified. It was composed of a single polypeptide chain migrating in SDS gels according to an Mr of 34,000. As in Salmonella typhimurium, the enzyme was associated in a bifunctional complex with O-acetylserine (thiol)-lyase. 相似文献
8.
Production of cysteine for bacterial and plant biotechnology: application of cysteine feedback-insensitive isoforms of serine acetyltransferase 总被引:10,自引:0,他引:10
The first step of cysteine biosynthesis in bacteria and plants consists in the formation of O-acetylserine catalyzed by serine acetyltransferase (SAT). SAT is highly sensitive to feedback inhibition by cysteine as part of the regulatory circuit of cysteine biosynthesis und thus hampers over-expression and fermentation of cysteine in biotechnological production processes. Since plants contain multiple SAT isoforms with different cysteine feedback sensitivity, this resource was exploited to demonstrate the suitability of plant SATs for the production of cysteine in both bacteria and plants. Three new cDNAs encoding SATs were isolated from Nicotiana tabacum. The catalytic activity of SAT4 was insensitive up to 0.6 mM cysteine. Expression of SAT4 in a newly constructed Escherichia coli host strain without endogenous SAT activity yielded a significant accumulation of cysteine in the culture medium compared to expression of cysteine sensitive SATs in the same strain. The application of a similarly insensitive SAT isoform from A. thaliana demonstrated the suitability of this approach to increase cysteine levels in transgenic tobacco plants. 相似文献
9.
When growing in its native habitat, Thlaspi goesingense can hyperaccumulate 1.2% of its shoot dry weight as nickel. We reported previously that both constitutively elevated activity of serine acetyltransferase (SAT) and concentration of glutathione (GSH) are involved in the ability of T. goesingense to tolerate nickel. A feature of SAT is its feedback inhibition by L-cysteine. To understand the role of this regulation of SAT by Cys on GSH-mediated nickel tolerance in T. goesingense, we characterized the enzymatic properties of SATs from T. goesingense. We demonstrate that all three isoforms of SAT in T. goesingense are insensitive to inhibition by Cys. Further, two amino acids (proline and alanine) in the C-terminal region of the cytosolic SAT (SAT-c) from T. goesingense are responsible for converting the enzyme from a Cys-sensitive to a Cys-insensitive form. Furthermore, the Cys-insensitive isoform of SAT-c confers elevated resistance to nickel when expressed in Escherichia coli and Arabidopsis thaliana, supporting a role for altered regulation of SAT by Cys in nickel tolerance in T. goesingense. 相似文献
10.
Bradford C. Braden Barry A. Fields Roberto J. Poljak 《Journal of molecular recognition : JMR》1995,8(5):317-325
The solvation of the antibody–antigen Fv D1.3–lysozyme complex is investigated through a study of the conservation of water molecules in crystal structures of the wild-type Fv fragment of antibody D1.3, 5 free lysozyme, the wild-type Fv D1.3–lysozyme complex, 5 Fv D1.3 mutants complexed with lysozyme and the crystal structure of an idiotope (Fv D1.3)-abti-idiotope (Fv E5.2) complex. In all, there are 99 water molecules common to the wild-type and mutant antibody–lysozyme complexes. The antibody–lysozyme interface includes 25 well-ordered solvent molecules, conserved among the wild-type and mutant Fv D1.3–lysozyme complexes, which are bound directly or through other water molecules to both antibody and antigen. In addition to contributing hydrogen bonds to the antibody–antigen interaction the solvent molecules fill many interface cavities. Comparison with x-ray crystal structures of free Fv D1.3 and free lysozyme shows that 20 of these conserved interface waters in the complex were bound to one of the free proteins. Uo to 23 additional water molecules are also found in the antibody–antigen interface, however these waters do no bridge antibody and antigen and their temperature factors are much higher than those of the 25 well-ordered waters. Fifteen water molecules are displaced to form the complex, some of which are substituted by hydrophilic protein atoms, and 5 water molecules are added at the antibody–antigen interface with the formation of the complex. While the current crystal models of the D1.3–lysozyme complex do not demonstrate the increase in bound waters found in a physico-chemical study of the interaction at decreased water activities, the 25 well-ordered interface water contribute a net gain of 10 hydrogen bonds to complex stability. 相似文献
11.
Nina Franko Konstantinos Grammatoglou Gabriele Costantino Aigars Jirgensons Andrea Mozzarelli 《Journal of enzyme inhibition and medicinal chemistry》2018,33(1):1343-1351
O-acetylserine sulfhydrylase (OASS) is the pyridoxal 5′-phosphate dependent enzyme that catalyses the formation of L-cysteine in bacteria and plants. Its inactivation is pursued as a strategy for the identification of novel antibiotics that, targeting dispensable proteins, holds a great promise for circumventing resistance development. In the present study, we have investigated the reactivity of Salmonella enterica serovar Typhimurium OASS-A and OASS-B isozymes with fluoroalanine derivatives. Monofluoroalanine reacts with OASS-A and OASS-B forming either a stable or a metastable α-aminoacrylate Schiff’s base, respectively, as proved by spectral changes. This finding indicates that monofluoroalanine is a substrate analogue, as previously found for other beta-halogenalanine derivatives. Trifluoroalanine caused different and time-dependent absorbance and fluorescence spectral changes for the two isozymes and is associated with irreversible inhibition. The time course of enzyme inactivation was found to be characterised by a biphasic behaviour. Partially distinct inactivation mechanisms for OASS-A and OASS-B are proposed. 相似文献
12.
GABA-TRANSAMINASES OF HUMAN BRAIN AND PERIPHERAL TISSUES—KINETIC AND MOLECULAR PROPERTIES 总被引:2,自引:2,他引:0
Abstract— Kinetic experiments with 4-aminobutyrate-2-ketoglutarate transaminase (GABA-T), partially purified from human brain tissue, supported a Bi Bi Ping-Pong type of enzyme mechanism in which the enzyme oscillates between forms bound to pyridoxal phosphate and pyridoxamine phosphate. Extrapolated K m values were 0.31 m m for γ-aminobutyrate, 0.16 m m for α-ketoglutarate, and 3.8 μ m for pyridoxal phosphate. Very similar kinetic parameters were observed with rat brain enzyme. Apparent molecular weight of human GABA-T by gel filtration was 70,000 ± 3000. Electrofucusing experiments indicated a single ionic form with isoelectric pH = 5.7. Enzyme activity was inhibited by Tris, halides, cadmium and cupric ions, and known GABA-T inhibitors.
GABA-transaminating enzymes isolated from human kidney and liver were found to be similar to the brain enzyme with respect to substrate affinities, cofactor requirements, isoelectric pH values, molecular weights, and response to inhibitors. 相似文献
GABA-transaminating enzymes isolated from human kidney and liver were found to be similar to the brain enzyme with respect to substrate affinities, cofactor requirements, isoelectric pH values, molecular weights, and response to inhibitors. 相似文献
13.
Interaction of purified human liver and placental alkaline phosphatases (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) with sulfhydryl groups, sulfhydryl reagents, and Mg2+ were studied. L-Cysteine (0.1 mmol/l) or Mg2+ activated the liver enzyme 4-5-fold and the placental enzyme 2-3-fold, with optimal pH 7.5-8.0; these activations were not additive. L-Cysteine (2 mmol/l) inhibited both enzymes maximally at pH greater than 9.0; phosphate protected the enzymes. S-Methylcysteine had little effect, with or without Mg2+. Inhibition by sulfur-containing compounds paralleled their ability to bind Zn2+. Fluoresceine mercury acetate (specific for sulfhydryl groups) inhibited the isoenzymes, whereas iodoacetic acid, iodoacetamide, dithionitrobenzoic acid, and p-chloromercuribenzoate had little effect. The inhibition was reversed by L-cysteine and only slightly protected by inorganic phosphate. Thus, there are two sites on human liver and placental alkaline phosphatase that interact with L-cysteine; a Mg2+-binding site, which results in activation, and a site that involves one or both of the bound Zn2+ ions and results in inactivation. Both enzymes have a protected essential thiol group. 相似文献
14.
有机相中脂肪酶催化不对称酯合成反应动力学的研究杨红,高修功,郭妮妮,曹淑桂,杨同书(吉林大学酶工程国家重点实验室,长春130023)在环已烷中应用酵母脂肪酶催化外消旋2-辛醇和辛酸的不对称酯合成反应,研究了该反应的动力学机制,测定了表现动力学常数.利... 相似文献
15.
Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme. 相似文献
16.
The kinetics of stereoselective esterification of racemic Naproxen with trimethylsilyl methanol by Candida cylindracea lipase in organic solvents has been investigated. A Ping-Pong Bi Bi mechanism with competitive inhibition by this alcohol for each enantiomer -has been identified. The rate equations were further analyzed in the time-course reaction after considering the effect of enzyme deactivation in the organic mixtures, but not in isooctane. Effects of the hydrophobicity of solvent on the solubility of the racemate, the kinetic parameters and their combinations are also discussed. 相似文献
17.
Markus Wirtz Hannah Birke Corinna Heeg Christopher Müller Fabian Hosp Christian Throm Stephan K?nig Anna Feldman-Salit Karsten Rippe Gabriele Petersen Rebecca C. Wade Vladimir Rybin Klaus Scheffzek Rüdiger Hell 《The Journal of biological chemistry》2010,285(43):32810-32817
Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = −33 kcal mol−1) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (KD = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (KD) and the regulation of cysteine sensitivity of SAT within the CSC. 相似文献
18.
Purification and properties of a new S-adenosyl-L-methionine:flavonoid 4'-O-methyltransferase from carnation (Dianthus caryophyllus L.). 总被引:1,自引:0,他引:1
Paolo Curir Virginia Lanzotti Marcello Dolci Paola Dolci Carlo Pasini Gordon Tollin 《European journal of biochemistry》2003,270(16):3422-3431
A new enzyme, S-adenosyl-l-methionine:flavonoid 4'-O-methyltransferase (EC 2.1.1.-) (F 4'-OMT), has been purified 1 399-fold from the tissues of carnation (Dianthus caryophyllus L). The enzyme, with a molecular mass of 43-45 kDa and a pI of 4.15, specifically methylates the hydroxy substituent in 4'-position of the flavones, flavanones and isoflavones in the presence of S-adenosyl-l-methionine. A high affinity for the flavone kaempferol was observed (Km = 1.7 micro m; Vmax = 95.2 micro mol.min-1.mg-1), while other 4'-hydroxylated flavonoids proved likewise to be suitable substrates. Enzyme activity had no apparent Mg++ requirement but was inhibited by SH-group reagents. The optimum pH value for F 4'-OMT activity was found to be around neutrality. Kinetic analysis of the enzyme bi-substrate reaction indicates a Ping-Pong mechanism and excludes the formation of a ternary complex. The F 4'-OMT activity was increased, in both in vitro and in vivo carnation tissues, by the inoculation with Fusarium oxysporum f. sp. dianthi. The enzyme did not display activity towards hydroxycinnamic acid derivatives, some of which are involved, as methylated monolignols, in lignin biosynthesis; the role of this enzyme could be therefore mainly defensive, rather than structural, although its precise function still needs to be ascertained. 相似文献
19.
Immobilization of Aspergillus oryzae tannase and properties of the immobilized enzyme 总被引:1,自引:0,他引:1
M. A. Abdel-Naby A. A. Sherif A. B. El-Tanash A. T. Mankarios 《Journal of applied microbiology》1999,87(1):108-114
Tannase enzyme from Aspergillus oryzae was immobilized on various carriers by different methods. The immobilized enzyme on chitosan with a bifunctional agent (glutaraldehyde) had the highest activity. The catalytic properties and stability of the immobilized tannase were compared with the corresponding free enzyme. The bound enzyme retained 20·3% of the original specific activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme. The optimum temperature of the reaction was determined to be 40 °C for the free enzyme and 55 °C for the immobilized form. The stability at low pH, as well as thermal stability, were significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent Km value and a lower energy of activation. The immobilized enzyme retained about 85% of the initial catalytic activity, even after being used 17 times. 相似文献
20.
Plants and bacteria assimilate and incorporate inorganic sulfur into organic compounds such as the amino acid cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase (CS) complex. The CS complex is composed of the enzymes serine acetyl transferase (SAT) and O-acetyl-serine-(thiol)-lyase (OAS-TL). Although it is experimentally known that formation of the CS complex influences cysteine production, the exact biological function of the CS complex, the mechanism of reciprocal regulation of the constituent enzymes and the structure of the complex are still poorly understood. Here, we used docking techniques to construct a model of the CS complex from mitochondrial Arabidopsis thaliana. The three-dimensional structures of the enzymes were modeled by comparative techniques. The C-termini of SAT, missing in the template structures but crucial for CS formation, were modeled de novo. Diffusional encounter complexes of SAT and OAS-TL were generated by rigid-body Brownian dynamics simulation. By incorporating experimental constraints during Brownian dynamics simulation, we identified complexes consistent with experiments. Selected encounter complexes were refined by molecular dynamics simulation to generate structures of bound complexes. We found that although a stoichiometric ratio of six OAS-TL dimers to one SAT hexamer in the CS complex is geometrically possible, binding energy calculations suggest that, consistent with experiments, a ratio of only two OAS-TL dimers to one SAT hexamer is more likely. Computational mutagenesis of residues in OAS-TL that are experimentally significant for CS formation hindered the association of the enzymes due to a less-favorable electrostatic binding free energy. Since the enzymes from A. thaliana were expressed in Escherichia coli, the cross-species binding of SAT and OAS-TL from E. coli and A. thaliana was explored. The results showed that reduced cysteine production might be due to a cross-binding of A. thaliana OAS-TL with E. coli SAT. The proposed models of the enzymes and their complexes provide mechanistic insights into CS complexation. 相似文献