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1.
Phillips RS Demidkina TV Zakomirdina LN Bruno S Ronda L Mozzarelli A 《The Journal of biological chemistry》2002,277(24):21592-21597
The binding of substrates and inhibitors to wild-type Proteus vulgaris tryptophan indole-lyase and to wild type and Y71F Citrobacter freundii tyrosine phenol-lyase was investigated in the crystalline state by polarized absorption microspectrophotometry. Oxindolyl-lalanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 microm, approximately 10-fold higher than that in solution. l-Trp or l-Ser react with tryptophan indole-lyase crystals to give, as in solution, a mixture of external aldimine and quinonoid intermediates and gem-diamine and external aldimine intermediates, respectively. Different from previous solution studies (Phillips, R. S., Sundararju, B., & Faleev, N. G. (2000) J. Am. Chem. Soc. 122, 1008-1114), the reaction of benzimidazole and l-Trp or l-Ser with tryptophan indole-lyase crystals does not result in the formation of an alpha-aminoacrylate intermediate, suggesting that the crystal lattice might prevent a ligand-induced conformational change associated with this catalytic step. Wild-type tyrosine phenol-lyase crystals bind l-Met and l-Phe to form mixtures of external aldimine and quinonoid intermediates as in solution. A stable quinonoid intermediate with lambda(max) at 502 nm is accumulated in the reaction of crystals of Y71F tyrosine phenol-lyase, an inactive mutant, with 3-F-l-Tyr with a dissociation constant of 1 mm, approximately 10-fold higher than that in solution. The stability exhibited by the quinonoid intermediates formed both by wild-type tryptophan indole-lyase and by wild type and Y71F tyrosine phenol-lyase crystals demonstrates that they are suitable for structural determination by x-ray crystallography, thus allowing the elucidation of a key species of pyridoxal 5'-phosphate-dependent enzyme catalysis. 相似文献
2.
Nikita A. Kuznetsov Nicolai G. Faleev Alexandra A. Kuznetsova Elena A. Morozova Svetlana V. Revtovich Natalya V. Anufrieva Alexei D. Nikulin Olga S. Fedorova Tatyana V. Demidkina 《The Journal of biological chemistry》2015,290(1):671-681
Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation. 相似文献
3.
Kulikova VV Zakomirdina LN Dementieva IS Phillips RS Gollnick PD Demidkina TV Faleev NG 《Biochimica et biophysica acta》2006,1764(4):750-757
Tyr72 is located at the active site of tryptophanase (Trpase) from Proteus vulgaris. For the wild-type Trpase Tyr72 might be considered as the general acid catalyst at the stage of elimination of the leaving groups. The replacement of Tyr72 by Phe leads to a decrease in activity for L-tryptophan by 50,000-fold and to a considerable rearrangement of the active site of Trpase. This rearrangement leads to an increase of room around the alpha-C atom of any bound amino acid, such that covalent binding of alpha-methyl-substituted amino acids becomes possible (which cannot be realized in wild-type Trpase). The changes in reactivities of S-alkyl-L-cysteines provide evidence for an increase of congestion in the proximity of their side groups in the mutant enzyme as compared to wild-type enzyme. The observed alteration of catalytic properties in a large degree originates from a conformational change in the active site. The Y72F Trpase retains significant activity for L-serine, which allowed us to conclude that in the mutant enzyme, some functional group is present which fulfills the role of the general acid catalyst in reactions associated with elimination of small leaving groups. 相似文献
4.
Melanoma is the most lethal cutaneous cancer with a highly aggressive and metastatic phenotype. While recent genetic and epigenetic studies have shed new insights into the mechanism of melanoma development, the involvement of regulatory non‐coding RNAs remain unclear. Long non‐coding RNAs (lncRNAs) are a group of endogenous non‐protein‐coding RNAs with the capacity to regulate gene expression at multiple levels. Recent evidences have shown that lncRNAs can regulate many cellular processes, such as cell proliferation, differentiation, migration and invasion. In the melanoma, deregulation of a number of lncRNAs, such as HOTAIR, MALAT1, BANCR, ANRIL, SPRY‐IT1 and SAMMSON, have been reported. Our review summarizes the functional role of lncRNAs in melanoma and their potential clinical application for diagnosis, prognostication and treatment. 相似文献
5.
Priscila R Moreira Marcos A Maioli Hyllana CD Medeiros Marieli Guelfi Flávia TV Pereira Fábio E Mingatto 《Biological research》2014,47(1)
Background
The liver is an important organ for its ability to transform xenobiotics, making the liver tissue a prime target for toxic substances. The carotenoid bixin present in annatto is an antioxidant that can protect cells and tissues against the deleterious effects of free radicals. In this study, we evaluated the protective effect of bixin on liver damage induced by carbon tetrachloride (CCl4) in rats.Results
The animals were divided into four groups with six rats in each group. CCl4 (0.125 mL kg-1 body wt.) was injected intraperitoneally, and bixin (5.0 mg kg-1 body wt.) was given by gavage 7 days before the CCl4 injection. Bixin prevented the liver damage caused by CCl4, as noted by the significant decrease in serum aminotransferases release. Bixin protected the liver against the oxidizing effects of CCl4 by preventing a decrease in glutathione reductase activity and the levels of reduced glutathione and NADPH. The peroxidation of membrane lipids and histopathological damage of the liver was significantly prevented by bixin treatment.Conclusion
Therefore, we can conclude that the protective effect of bixin against hepatotoxicity induced by CCl4 is related to the antioxidant activity of the compound. 相似文献6.
7.
Crystals of the apoenzyme of tyrosine phenol-lyase (EC 4.1.99.2), a pyridoxal 5'-phosphate-dependent enzyme from Citrobacter intermedius, have been grown by vapor diffusion of an ammonium sulfate solution to a protein solution. The crystals belong to space group P2(1)2(1)2, with dimensions of a = 75.5 A, b = 138.4 A and c = 94.1 A and diffract up to 2.7 A resolution. The asymmetric unit contains one half of the enzyme tetrameric molecule. Two heavy-atom derivatives of the crystals have been obtained. 相似文献
8.
Pioselli B Bettati S Demidkina TV Zakomirdina LN Phillips RS Mozzarelli A 《Protein science : a publication of the Protein Society》2004,13(4):913-924
The pyridoxal 5'-phosphate-dependent enzymes tyrosine phenol-lyase and tryptophan indole-lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the distinct spectral properties of catalytic intermediates and reaction products. The encapsulated enzymes exhibit altered distributions of ketoenamine and enolimine tautomers, increased values of inhibitors dissociation constants, slow attaining of steady-state in the presence of substrate and substrate analogs, modified steady-state distribution of catalytic intermediates, and a sixfold-eightfold decrease of specific activities. This behavior can be rationalized by a reduced conformational flexibility for the encapsulated enzymes and a selective stabilization of either the open (inactive) or the closed (active) form of the enzymes. Despite very similar structures and catalytic mechanisms, the influence of encapsulation is more pronounced for tyrosine phenol-lyase than tryptophan indole-lyase. This finding indicates that subtle structural and dynamic differences can lead to distinct interactions of the protein with the gel matrix. 相似文献
9.
pH dependencies of the main kinetic parameters for the reaction of tyrosine phenol-lyase (TPL) with L-tyrosine were studied earlier at pH from 6.0 to 9.5. At this range, L-tyrosine, whose pK(a) for the phenol hydroxyl is 10.5, exists as the zwitterion. It was concluded that zwitterion is the only "active" form for any tyrosine-like substrate. In the present work, we examined pH dependencies for 2-fluorotyrosine, 3-fluorotyrosine, 3,5-difluorotyrosine, 2,5-difluorotyrosine, 2,6-difluorotyrosine, and 3-chlorotyrosine which are more acidic than tyrosine. Respective pK(a)'s are 9.2, 8.7, 7.3, 7.9, 8,35, and 8.3. At higher pH, most of these substrates exist predominantly as anions, having two negative charges at the carboxylic and phenol groups, and one positive charge at the amino group. No decrease of k(cat)/K(m) values attributable to acidic dissociation of the phenol group was found. From comparison of theoretical curves with the experimental data, we conclude that most likely, both zwitterion and anion forms of 3-fluorotyrosine, 3,5-difluorotyrosine, 2,5-difluorotyrosine, 2,6-difluorotyrosine, and 3-chlorotyrosine may be bound and subsequently catalytically transformed by TPL. The reactivities of the two forms are quite comparable. The roles of catalytic groups in the active site, especially Arg381 and Thr-124, are discussed. 相似文献
10.
A gene encoding L-methionine gamma-lyase is present in Enterobacteriaceae family genomes: identification and characterization of Citrobacter freundii L-methionine gamma-lyase
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Manukhov IV Mamaeva DV Rastorguev SM Faleev NG Morozova EA Demidkina TV Zavilgelsky GB 《Journal of bacteriology》2005,187(11):3889-3893
Citrobacter freundii cells produce L-methionine gamma-lyase when grown on a medium containing L-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded L-methionine gamma-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3'-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found. 相似文献