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1.
A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibition of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thus inhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflammatory drugs. Structure–activities studies guided optimization of a lead compound, identified through high-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in human liver microsomal assay suitable for preclinical development. 相似文献
2.
Abstract Aspergillus niger possesses a galactofuranosidase activity, however, the corresponding enzyme or gene encoding this enzyme has never been identified. As evidence is mounting that enzymes exist with affinity for both arabinofuranose and galactofuranose, we investigated the possibility that α-l-arabinofuranosidases, encoded by the abfA and abfB genes, are responsible for the galactofuranosidase activity of A. niger. Characterization of the recombinant AbfA and AbfB proteins revealed that both enzymes do not only hydrolyze p-nitrophenyl-α-l-arabinofuranoside (pNp-α-Araf) but are also capable of hydrolyzing p-nitrophenyl-β-d-galactofuranoside (pNp-β-Galf). Molecular modeling of the AbfB protein with pNp-β-Galf confirmed the possibility for AbfB to interact with this substrate, similarly as with pNp-α-Araf. We also show that galactomannan, a cell wall compound of A. niger, containing β-linked terminal and internal galactofuranosyl moieties, can be degraded by an enzyme activity that is present in the supernatant of inulin-grown A. niger. Interestingly, purified AbfA and AbfB did not show this hydrolyzing activity toward A. nigergalactomannan. In summary, our studies demonstrate that AbfA and AbfB, α-l-arabinofuranosidases from different families, both contain a galactofuranose (Galf)-hydrolyzing activity. In addition, our data support the presence of a Galf-hydrolase activity expressed by A. niger that is capable of degrading fungal galactomannan. 相似文献
3.
We investigated N-adamantyl- N′-phenyl urea derivatives as simple sEH inhibitors. Salicylate ester derivatives have high inhibitory activities against human sEH, while the free benzoic acids are less active. The methyl salicylate derivative is a potent sEH inhibitor, which also has high metabolic and chemical stabilities; suggesting that such inhibitors are potential lead molecule for bioactive compounds acting in vivo. 相似文献
5.
1. The nature of the acetyl-CoA hydrolase (EC 3.1.2.1) reaction in rat and sheep liver homogenates was investigated. 2. The activity determined in an incubated system was 5.10 and 3.28nmol/min per mg of protein for rat and sheep liver homogenate respectively. This activity was not affected by the addition of l-carnitine, but was decreased by the addition of d-carnitine. 3. No acetyl-CoA hydrolase activity could be detected in rat or sheep liver homogenates first treated with Sephadex G-25. This treatment decreased the carnitine concentrations of the homogenates to about one-twentieth. Subsequent addition of l-carnitine, but not d-carnitine, restored the apparent acetyl-CoA hydrolase activity. 4. Sephadex treatment did not affect acetyl-carnitine hydrolase activity of the homogenates, which was 5.8 and 8.1nmol/min per mg of protein respectively for rat and sheep liver. 5. Direct spectrophotometric assay of acetyl-CoA hydrolase, based on the reaction of CoA released with 5,5'-dithiobis-(2-nitrobenzoic acid), clearly demonstrated that after Sephadex treatment no activity could be measured. 6. Carnitine acetyltransferase (EC 2.3.1.7) activity measured in the same assay system in response to added l-carnitine was very low in normal rat liver homogenates, owing to the apparent high acetyl-CoA hydrolase activity, but was increased markedly after Sephadex treatment. The V(max.) for this enzyme in rat liver homogenates was increased from 3.4 to 14.8nmol/min per mg of protein whereas the K(m) for l-carnitine was decreased from 936 to 32mum after Sephadex treatment. 7. Acetyl-CoA hydrolase activity could be demonstrated in disrupted rat liver mitochondria but not in separated outer or inner mitochondrial membrane fractions. Activity could be demonstrated after recombination of outer and inner mitochondrial membrane fractions. The outer mitochondrial membrane fraction showed acetylcarnitine hydrolase activity and the inner mitochondrial membrane fraction showed carnitine acetyltransferase activity. 8. The results presented here demonstrate that acetyl-CoA hydrolase activity in rat and sheep liver is an artifact and the activity is due to the combined activity of carnitine acetyltransferase and acetylcarnitine hydrolase. 相似文献
6.
Bacteroides?thetaiotaomicron VPI-5482 harbors a gene encoding a putative cycloisomaltooligosaccharide glucanotransferase (BT3087) belonging to glycoside hydrolase family?66. The goal of the present study was to characterize the catalytic properties of this enzyme. Therefore, we expressed BT3087 (recombinant endo-dextranase from Bacteroides?thetaiotaomicron VPI-5482) in Escherichia?coli and determined that recombinant endo-dextranase from Bacteroides?thetaiotaomicron VPI-5482 preferentially synthesized isomaltotetraose and isomaltooligosaccharides (degree of polymerization >?4) from dextran. The enzyme also generated large cyclic isomaltooligosaccharides early in the reaction. We conclude that members of the glycoside hydrolase?66 family may be classified into three types: (a) endo-dextranases, (b) dextranases possessing weak cycloisomaltooligosaccharide glucanotransferase activity, and (c) cycloisomaltooligosaccharide glucanotransferases. 相似文献
7.
The hydrolysis of cellobiose by β-glucodisases is an important step of cellulose biodegradation. However, the interactive mechanism between cellobiose and β-glucosidases is still unclear until now. Thus, in this study, we explored the binding modes between cellobiose and three β-glucosidases from glycoside hydrolase family 1 by means of molecular docking. The three β-glucosidases were named as TmGH1 (from bacterium Thermotoga), SsGH1 (from archaea Sulfolobus solfataricus) and TrGH1 (from fungus Trichoderma reesei) respectively, according to the monophyletic groups they belong to. Molecular dockings were performed between cellobiose and the three β-glucosidases, resulting in three optimum docking complexes, that is TmGH1-cellobiose, SsGH1-cellobiose and TrGh1-cellobiose complexes. Our docking results indicated that there were non-bonded interactions between cellobiose and the three β-glucosidases. The binding affinities of the three complexes were -13.6669kJ/mol, -13.2973kJ/mol and -18.6492kJ/mol, respectively. Then the detailed interactions were investigated, which revealed the key amino acid residues interacted with cellobiose by hydrogen bonds (H-bonds) or hydrophobic interactions. It was observed that most of the key residues involved in the non-bonded interactions were equivalent and conserved for the three complexes, and these residues were a glutamine, a histidine, a tyrosine, a phenylalanine, three glutamics, and four tryptophans. This information is of great importance for designing β-glucosidase with higher cellobiose-hydrolyzing efficiency. 相似文献
8.
γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways. Electronic supplementary materialThe online version of this article (doi:10.1007/s12263-014-0444-0) contains supplementary material, which is available to authorized users. 相似文献
9.
We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower. 相似文献
10.
The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. β-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active β-xylosidases, some of which could have practical applications. The biochemical details of four GH43 β-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers ( B. pumilus) or mixtures of homodimers and homotetramers ( B. subtilis and L. brevis). k cat and k cat/ K m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 β-xylosidases. The K i values for d-glucose (83.3–357 mM) and d-xylose (15.6–70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature ( K t 0.5?~?45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k cat and k cat/ K m on natural substrates xylobiose (407 s ?1, 138 s ?1?mM ?1), xylotriose (235 s ?1, 80.8 s ?1?mM ?1), and xylotetraose (146 s ?1, 32.6 s ?1?mM ?1). 相似文献
11.
We obtained Cx1 from a commercial supplier, whose catalog listed it as a β-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein sequencing indicates Cx1 is similar to but not identical to β-xylosidases of GH52, including Q09LZ0, that have retaining mechanisms. Q09LZ0 β-xylosidase had been characterized biochemically in kinetic reactions that contained Tris. We overproduced Q09LZ0 and demonstrated that Tris is a competitive inhibitor of the β-xylosidase. Also, the previous work used grossly incorrect extinction coefficients for product 4-nitrophenol. We redetermined kinetic parameters using reactions that omitted Tris and using correct extinction coefficients for 4-nitrophenol. Cx1 and Q09LZ0 β-xylosidases were thus shown to possess similar kinetic properties when acting on 4-nitrophenyl-β-d-xylopyranoside and xylobiose. kcat pH profiles of Cx1 and Q09LZ0 acting on 4-nitrophenyl-β-d-xylopyranoside and xylobiose have patterns containing two rate increases with increasing acidity, not reported before for glycoside hydrolases. The dexylosylation step of 4-nitrophenyl-β-d-xylopyranoside hydrolysis mediated by Q09LZ0 is not rate determining for kcat4NPX. 相似文献
12.
Incorporation of an adamantyl group in prototypical soluble expoxide hydrolase (sEH) inhibitors afforded improved enzyme potency. We explored replacement of the adamantyl group in unsymmetrical ureas and amides with substituted aryl rings to identify equipotent and metabolically stable sEH inhibitors. We found that aryl rings, especially those substituted in the para position with a strongly electron withdrawing substituent, afforded enzyme IC 50 values comparable to the adamantyl compounds in an ether substituted, unsymmetrical N, N′-diaryl urea or amide scaffold. 相似文献
13.
β-N-Acetylglucosaminidases (GlcNAcases) are important for many biological functions and industrial applications. In this study, a glycoside hydrolase family 20 GlcNAcase from Shinella sp. JB10 was expressed in Escherichia coli BL21 (DE3). Compared to many GlcNAcases, the purified recombinant enzyme (rJB10Nag) exhibited a higher specificity activity (538.8 µmol min−1 mg−1) or V
max (1030.0 ± 82.1 µmol min−1 mg−1) toward p-nitrophenyl β-N-acetylglucosaminide and N,N′-diacetylchitobiose (specificity activity of 35.4 µmol min−1 mg−1) and a higher N-acetylglucosaminide tolerance (approximately 50% activity in 70.0 mM N-acetylglucosaminide). The degree of synergy on enzymatic degradation of chitin by a commercial chitinase and rJB10Nag was as high as 2.35. The enzyme was tolerant to most salts, especially 3.0–15.0% (w/v) NaCl and KCl. These biochemical characteristics make the JB10 GlcNAcase a candidate for use in many potential applications, including processing marine materials and the bioconversion of chitin waste. Furthermore, the enzyme has the highest proportions of alanine (16.5%), glycine (10.5%), and random coils (48.8%) with the lowest proportion of α-helices (24.9%) among experimentally characterized GH 20 GlcNAcases from other organisms. 相似文献
14.
The present study compared the properties of cholesterol ester hydrolase(s) in myelin and microsomes from rat, mouse and human brain. The results indicated that the enzyme activity in both myelin and microsomes from rat, mouse and human brain was optimal at pH 6.5 and required Triton X-100 for optimal activity. The enzyme activity in myelin was 3- to 4-fold higher in the presence of Trition X-100 than taurocholate. Addition of phosphatidyl serine enhanced (2 to 4 fold) the hydrolase activity in both myelin and microsomes. The properties of the enzyme in solubilized preparation of myelin were also similar to the properties of the enzyme in partially delipidated and solubilized preparations of microsomes. The activity was again optimal at pH 6.5, required Triton X-100 for optimal activity and was stimulated by phosphatidyl serine. These results indicate that the properties of cholesterol ester hydrolase in myelin are similar to those of the microsomal enzyme and that this is true for the fractions from both human and rodent brain. The data thus lead us to believe that the hydrolase activity in mammalian brain myelin and microsomes may reflect the distribution of a single enzyme in the two fractions rather than two distinct enzymes, one being specific to each fraction. 相似文献
15.
Purification of a cis-epoxysuccinic acid hydrolase was achieved by ammonium sulfate precipitation, ionic exchange chromatography, hydrophobic interaction chromatography followed by size-exclusion chromatography. The enzyme was purified 177-fold with a yield of 14.4%. The apparent molecular mass of the enzyme was determined to be 33 kDa under denaturing conditions. The optimum pH for enzyme activity was 7.0, and the enzyme exhibited maximum activity at about 45 °C in 50 mM sodium phosphate buffer (pH 7.5). EDTA and o-phenanthrolin inhibited the enzyme activity remarkably, suggesting that the enzyme needs some metal cation to maintain its activity. Results of inductively coupled plasma mass spectrometry analysis indicated that the cis-epoxysuccinic acid hydrolase needs Zn 2+ as a cofactor. Eight amino acids sequenced from the N-terminal region of the cis-epoxysuccinic acid hydrolase showed the same sequence as the N-terminal region of the beta subunit of the cis-epoxysuccinic acid hydrolase obtained from Alcaligenes sp. 相似文献
16.
Previously, we identified two closely related proteins termed W14 and W15 that were enriched in the overwinter buds of the
gentian plant Gentiana triflora. Expression of the latter protein W15 has been implicated in its association with cold hardiness, because of its absence
in a cold-sensitive mutant. Here, we characterized these two proteins and the genes encoding them. Amino acid sequences of
the W14 and W15 proteins showed difference at only three amino acid positions, and both of them showed homologies to α/β hydrolase
fold superfamily. Consistently, GST-fused W14 and W15 proteins expressed in bacteria showed hydrolase activity toward 1-naphtyl
acetate. Structural analysis of these two genes in seven different gentian strains/cultivars including an anther culture-derived
homozygous diploid revealed that W14 and W15 genes are allelic. Three genotypes were found; two strains carried both alleles ( W14/ W15), one carried the W15 genes in both alleles ( W15/ W15), and others were homozygous of W14 ( W14/ W14). Interestingly, expression of the two proteins exhibited allele-specificity. In one W14/ W15 strain, expression of the W15 allele was almost repressed. In addition, organ specific expression of the alleles was observed in different cultivars. These
observations were discussed in relation to winter hardiness of the gentian plants.
Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB281493 and
AB281494. 相似文献
17.
Escherichia coli YicI is a retaining α-xylosidase, which strictly recognizes the α-xylosyl moiety at the non-reducing end, belonging to glycoside hydrolase family 31 (GH 31). We have elucidated key residues determining the substrate specificity at both glycone and aglycone sites of Escherichia coli α-xylosidase (YicI). Detection of distinguishing features between α-xylosidases and α-glucosidases of GH 31 in their close evolutionary relationship has been used for the modification of protein function, converting YicI into an α-glucosidase. Aglycone specificity has been characterized by its transxylosylation ability. YicI exhibits a preference for aldopyranosyl sugars having equatorial 4-OH as the acceptor substrate with 1,6 regioselectivity, resulting in transfer products. The disaccharide transfer products of YicI, α- d-Xyl p-(1→6)- d-Man p, α- d-Xyl p-(1→6)- d-Fru f, and α- d-Xyl p-(1→3)- d-Fru p, are novel oligosaccharides, which have never been reported. The transxylosylation products are moderately inhibitory towards intestinal α-glucosidases. 相似文献
19.
Background Glycosyl hydrolase family 1 (GH1) β-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 β-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice. Results Forty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed β-(1,4)-linked oligosaccharides of 3–6 glucose residues and laminaribiose. Conclusion Careful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 β-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism. 相似文献
20.
β-Xylosidases are hemicellulases that hydrolyze short xylo-oligosaccharides into xylose units, thus complementing endoxylanase degradation of the hemicellulose component of lignocellulosic substrates. Here, we describe the cloning, characterization, and kinetic analysis of a glycoside hydrolase family 43 β-xylosidase (Xyl43A) from the aerobic cellulolytic bacterium, Thermobifida fusca. Temperature and pH optima of 55-60 °C and 5.5-6, respectively, were determined. The apparent K(m) value was 0.55 mM, using p-nitrophenyl xylopyranoside as substrate, and the catalytic constant (k(cat)) was 6.72 s(-1). T. fusca Xyl43A contains a catalytic module at the N terminus and an ancillary module (termed herein as Module-A) of undefined function at the C terminus. We expressed the two recombinant modules independently in Escherichia coli and examined their remaining catalytic activity and binding properties. The separation of the two Xyl43A modules caused the complete loss of enzymatic activity, whereas potent binding to xylan was fully maintained in the catalytic module and partially in the ancillary Module-A. Nondenaturing gel electrophoresis revealed a specific noncovalent coupling of the two modules, thereby restoring enzymatic activity to 66.7% (relative to the wild-type enzyme). Module-A contributes a phenylalanine residue that functions as an essential part of the active site, and the two juxtaposed modules function as a single functional entity. 相似文献
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