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排序方式: 共有589条查询结果,搜索用时 46 毫秒
91.
92.
Park SH Park HY Sohng JK Lee HC Liou K Yoon YJ Kim BG 《Biotechnology and bioengineering》2009,102(4):988-994
Glycosyltransferases (GTs) are crucial enzymes in the biosynthesis and diversification of therapeutically important natural products, and the majority of them belong to the GT-B superfamily, which is composed of separate N- and C-domains that are responsible for the recognition of the sugar acceptor and donor, respectively. In an effort to expand the substrate specificity of GT, a chimeric library with different crossover points was constructed between the N-terminal fragments of kanamycin GT (kanF) and the C-terminal fragments of vancomycin GT (gtfE) genes by incremental truncation method. A plate-based pH color assay was newly developed for the selection of functional domain-swapped GTs, and a mutant (HMT31) with a crossover point (N-kanF-669 bp and 753 bp-gtfE-C) for domain swapping was screened. The most active mutant HMT31 (50 kDa) efficiently catalyzed 2-DOS (aglycone substrate for KanF) glucosylation using dTDP-glucose (glycone substrate for GtfE) with k(cat)/K(m) of 162.8 +/- 0.1 mM(-1) min(-1). Moreover, HMT31 showed improved substrate specificity toward seven more NDP-sugars. This study presents a domain swapping method as a potential means to glycorandomization toward various syntheses of 2-DOS-based aminoglycoside derivatives. 相似文献
93.
Dravidum Maitraie Chi-Feng Hung Huang-Yao Tu Ya-Ting Liou Bai-Luh Wei Shyh-Chyun Yang Jih-Pyang Wang Chun-Nan Lin 《Bioorganic & medicinal chemistry》2009,17(7):2785-2792
Twenty 18β-glycyrrhetic acid (18β-GA) derivatives 2–21 including 13 new 18β-GA derivatives were synthesized and evaluated as anti-inflammatory and antioxidant agents. Compounds 7 and 20 with a 3,4-seco-structure and compound 6 with a lactone moiety showed potent inhibitory effect on superoxide anion generation in rat neutrophils response to fMLP/CB and PMA, respectively. Compound 6 with a lactone moiety revealed stronger inhibitory effect on XO activity than those of compounds 13 and 14 with a 3,4-seco-struture. Compound 14, a 30-isoproylcarbamoyl seco-compound exhibited potent inhibitory effect on NO accumulation and iNOS protein expression while compounds 3, 10, 13, 15, 17, and 21 revealed potent inhibitory effect on tumor necrosis factor-α (TNF-α) formation in RAW 264.7 cells in response to lipopolysaccharide (LPS). The cleavage of ring A of 3 attenuated the inhibitory effect on TNF-α formation in RAW 264.7 cells in response to LPS except for 17. The present results suggested these compounds were potential to be served as anti-inflammatory and antioxidant agents. 相似文献
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Aims: Paromamine is a vital and common intermediate in the biosynthesis of 4,5 and 4,6‐disubstituted 2‐deoxystreptamine (DOS)‐containing aminoglycosides. Our aim is to develop an engineered Escherichia coli system for heterologous production of paromamine. Methods and Results: We have constructed a mutant of E. coli BL21 (DE3) by disrupting glucose‐6‐phosphate isomerase (pgi) of primary metabolic pathway to increase glucose‐6‐phosphate pool inside the host. Disruption was carried out by λ Red/ET recombination following the protocol mentioned in the kit. Recombinants bearing 2‐deoxy‐scyllo‐inosose (DOI), DOS and paromamine producing genes were constructed from butirosin gene cluster and heterologously expressed in engineered host designed as E. coli BL21 (DE3) Δpgi. Secondary metabolites produced by the recombinants fermentated in 2YTG medium were extracted, and analysis of the extracts showed there is formation of DOI, DOS and paromamine. Conclusions: Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside biosynthesis. Significance and Impact of the Study: This is the first report of heterologous expression of paromamine gene set in E. coli. Hence a new platform is established in E. coli system for the production of paromamine which is useful for the exploration of novel aminoglycosides by combinatorial biosynthesis of 4,5‐ and 4,6‐disubtituted route of DOS‐containing aminoglycosides. 相似文献
96.
Liou B Kazimierczuk A Zhang M Scott CR Hegde RS Grabowski GA 《The Journal of biological chemistry》2006,281(7):4242-4253
Acid beta-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo-beta-glucosidase whose defective activity leads to the Gaucher disease phenotypes. To move toward a structure/function map for disease mutations, 52 selected single amino acid substitutions were introduced into GCase, expressed in an insect cell system, purified, and characterized for basic kinetic, stability, and activator response properties. The variant GCases from Gaucher disease patients and selected variant GCases from the mouse had decreased relative k(cat) and differential effects on active site binding and/or attachment of mechanism-based covalent (conduritol B epoxide) or reversible (deoxynojirimycin derivatives) inhibitors. A defect in negatively charged phospholipid activation was present in the majority of variant GCases but was increased in two, N370S and V394L. Deficits in saposin C enhancement of k(cat) were present in variant GCases involving residues 48-122, whereas approximately 2-fold increases were obtained with the L264I GCase. About 50% of variant GCases each had wild-type or increased sensitivity to in vitro cathepsin D digestion. Mapping of these properties onto the crystal structures of GCase indicated wide dispersion of functional properties that can affect catalytic function and stability. Site-directed mutagenesis of cysteine residues showed that the disulfide bonds, Cys(4)-Cys(16) and Cys(18)-Cys(23), and a free Cys(342) were essential for activity; the free Cys(126) and Cys(248) were not. Relative k(cat) was highly sensitive to a His substitution at Arg(496) but not at Arg(495). These studies and high phylogenetic conservation indicate localized and general structural effects of Gaucher disease mutations that were not obvious from the nature of the amino acid substitution, including those predicted to be nondisruptive (e.g. Val --> Leu). These results provide initial studies for the engineering of variant GCases and, potentially, molecular chaperones for therapeutic use. 相似文献
97.
Exposure to sublethal stress can trigger endogenous protection against subsequent, higher levels of stress. We tested for this preconditioning phenomenon in a model of Parkinson's disease by applying 6-hydroxydopamine to the dopaminergic MN9D cell line. Exposure to sublethal concentrations of 6-hydroxydopamine (5-10 microM) protected against the toxic effects of a subsequent exposure to a higher concentration (50 microM), as measured by the Hoechst assay for nuclear viability. This was accompanied by little or no protection against 6-hydroxydopamine-induced lactate dehydrogenase release, decline in ATP, or reduction in (3)H-dopamine uptake. The antioxidant, N-acetyl cysteine (20 mM), when applied during preconditioning, abolished protection, as did the protein synthesis inhibitor, cycloheximide (0.2 microM). Preconditioning did not affect superoxide dismutase or glutathione peroxidase enzymes, or levels of heat shock protein-72. However, Bcl-2 protein levels rose with preconditioning. Preconditioning rapidly increased phosphorylation of kinases ERK1/2, Akt and JNK, and was abolished by pharmacological inhibitors of their activity. Finally, sublethal 6-hydroxydopamine preconditioned against the toxicity of proteasome inhibitor, MG-132 (1 microM). Thus, exposure of a dopaminergic cell line to sublethal oxidative stress can protect against additional oxidative stress due to translational and post-translational modifications, as well as confer 'cross-tolerance' against a different insult, proteasome inhibition. 相似文献
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Pitts SL Liou GF Mitchenall LA Burgin AB Maxwell A Neuman KC Osheroff N 《Nucleic acids research》2011,39(11):4808-4817
It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3'-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme. 相似文献