Study on a novel dehydrochlorinase from BHC-degrading bacterium
一种新型BHC降解菌脱氯化氢酶的研究
基本信息
- 批准号:11660075
- 负责人:
- 金额:$ 2.3万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (C)
- 财政年份:1999
- 资助国家:日本
- 起止时间:1999 至 2000
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
γ-Hexachlorocyclohexane dehydrochlorinase (LinA) is a unique dehydrochlorinase that has no homologous sequence at the amino acid-sequence level, and for which the evolutionary origin is unknown. We here propose that LinA is a member of a novel structural superfamily of enzymes containing scytalone dehydratase, 3-oxo-Δ5-steroid isomerase, nuclear transport factor-2, and the β-subunit of naphthalene dioxygenase-all known structures with different functions. The three-dimensional structure of LinA is predicted by homology modeling. More than twenty mutants were prepared by site-directed mutagenesis. These mutants were expressed in E.coli, and their activities in crude extract were evaluated. Most of the features of the LinA mutants could be explained on the basis of the present LinA model, indicating its validity. We conclude that LinA catalyses the proton abstraction via the catalytic dyad H73-D25 by the similar mechanism as described for scytalone dehydratase. The results suggest that LinA and scytalone dehydratase evolved from a common ancestor. LinA may have evolved from an enzyme showing a dehydratase activity.Stereochemical analysis of the reaction products formed during conversion of γ-HCH by LinA was investigated by GC-MS, NMR, CD and molecular modeling. LinA requires the presence of a 1, 2-biaxial HCl pair on a substrate molecule. LinA enantiotopologically differentiates two 1, 2-biaxial HCl pairs present on γ-HCH and gives rise to a single PCCH enantiomer 1, 3 (R), 4 (S), 5(S), 6 (R)-PCCH.Furthermore, LinA enantiomerically differentiates 1, 3 (S), 4 (R), 5 (R), 6 (S)-PCCH and 1, 3 (R), 4 (S), 5 (S), 6 (R)-PCCH.The proposed mechanism of enzymatic biotransformation of γ-HCH to 1, 2, 4-TCB by LinA consists of two 1, 2-anti conformationally dependent dehydrochlorinations followed by 1, 4-anti dehydrochlorination.
γ-六氯环己烷脱氯化氢酶(LinA)是一种独特的脱氯化氢酶,在氨基酸水平上没有同源序列,其进化起源尚不清楚。我们在这里提出,LinA是一个新的结构超家族的酶,包括scytalone酶,3-氧代-Δ5-类固醇异构酶,核转运因子-2,和萘双加氧酶的β-亚基-所有已知的结构与不同的功能。通过同源模建预测了LinA的三维结构。通过定点突变制备了二十多个突变体。这些突变体在大肠杆菌中表达,并在粗提物中评价其活性。LinA突变体的大部分特征都可以用该模型来解释,表明该模型是有效的。我们的结论是,LinA催化质子提取通过催化二联体H73-D25的类似机制所描述的scytalone酶。结果表明,LinA和scytalone酶从一个共同的祖先进化而来。采用GC-MS、NMR、CD和分子模拟等方法对LinA转化γ-HCH的反应产物进行了立体化学分析。LinA需要在底物分子上存在1,2-双轴HCl对。LinA对映异构体区分了γ-HCH上的两个1,2-双轴HCl对,并产生单一的PCCH对映体1,3(R),4(S),5(S),6(R)-PCCH。6(R)-PCCH. LinA将γ-HCH酶促转化为1,2,4-TCB的机制包括两个1,2-反构象依赖性脱氯化氢和随后的1,4-反脱氯化氢。
项目成果
期刊论文数量(22)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Glatz, Z., M.V.Marini, M.Wimmerova, J.Damborsky, and Y.Nagata: "Determination of haloalkane dehalogenase activity by capillary zone electrophoresis."Journal of Chromatography A. 895. 219-225 (2000)
Glatz, Z.、M.V.Marini、M.Wimmerova、J.Damborsky 和 Y.Nagata:“通过毛细管区带电泳测定卤代烷脱卤酶活性。”色谱杂志 A. 895. 219-225 (2000)
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Marek, J., J.Vevodova, I.K.Smatanova, Y.Nagata, L.A.Svensson, J.Newman, M.Takagi, and J.Damborsky: "Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26."Biochemistry. 39. 14082-14086 (2000)
Marek, J.、J.Vevodova、I.K.Smatanova、Y.Nagata、L.A.Svensson、J.Newman、M.Takagi 和 J.Damborsky:“少动鞘氨醇单胞菌 UT26 的卤代烷脱卤酶的晶体结构。”生物化学。
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Y. Nagata ら: "Construction and characterization of histidine-tagged haloalkane dehalogenase (LinB) of a new substrate class from a ‥‥"Protein Expression and Purification. 17. 299-304 (1999)
Y. Nagata 等人:“来自 ‥‥ 的新底物类别的组氨酸标记卤代烷脱卤酶 (LinB) 的构建和表征”蛋白质表达和纯化 17. 299-304 (1999)。
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- 影响因子:0
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Nagata, Y., K.Miyauchi, and M.Takagi: "Complete analysis of genes and enzymes for γ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26."Journal of Industrial Microbiology & Biotechnology. 23. 380-390 (1999)
Nagata, Y.、K. Miyauchi 和 M. Takagi:“少动鞘氨醇单胞菌 UT26 中 γ-六氯环己烷降解的基因和酶的完整分析。”工业微生物学与生物技术杂志 23. 380-390 (1999)。
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- 影响因子:0
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Y. Nagata ら: "Complete analysis of genes and enzymes for γ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26"J. Industrial Microbiology & Biotechnology. 23. 380-390 (1999)
Y. Nagata 等:“少动鞘氨醇单胞菌 UT26 中 γ-六氯环己烷降解的基因和酶的完整分析”J.工业微生物学与生物技术。
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