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Research for the molecular evolution of the adaptive mutants obtained after continuous culture of cumene degrader on biphenyl.

联苯异丙苯降解剂连续培养获得的适应性突变体的分子进化研究。

基本信息

批准号：
08660089
负责人：
OMORI Toshio
金额：
$ 1.47万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08660089/
关键词：
cumene biphenyl Pseudomonas serine hydrolase adaptation Pseudomonas SP PCB

项目摘要

Cumene ( isopropylbenzene ) is an aromatic hydrocarbon with a chemical structure close to those of toluene and biphenyl. P.fluorescens IP01 can grow on cumene or toluene as a sole carbon and energy source but not on biphenyl. We surmised that the growth substrate specificity of strain IP01 is ascribed to the substrate specificity of the meta-cleavage compound hydrolase (CumD ).In this study, we attempted to determine the residue responsible for the subatrate specificity in CumD by means of site-directed mutagenesis. Amino acid sequence comparison between CumD and other hydrolases led us to identify several conserved residues likely to have a functional role in the catalytic center of CumD,that is, three amino acids, Ser^<103>, Asp^<224>, His^<252>, were found to be arranged in a sequential order similar to that in serine hydrolases. Substitutions of each of these three residues by Ala decreased enzymatic activity on hydrolisis of meta-cleavage compound of cumene (HOMODA ) below 5%. The … More se results suport the hypothesis that Ser^<103>, Asp^<224>, His^<252> constitute the catalytic triad of CumD.Substitution of Ile^<256> by Trp and that of Trp^<143> by Phe increased enzymatic activity on hydrolysis of meta-cleavage compound of biphenly ( HOPDA ). This result suggests that Ile^<256> and Trp^<143> are the key residues to alter the substrate specificity of CumD so as to hydolase HOPDA.It was also indicated that substitution of Val^<227> by Ile increased enzymatic activity on hydrolysis of HOMODA.This result suggests that Val^<227> is Iocated nearby the catalytic triad topologically, and is involved in the substrate-binding site in CumD.We obtained 25 mutants degrading efficiently biphenyl after continuous culture of IP01 on biphenyl containing a small amount of cumene. In these mutants, it was predicted that substrate specificity of CumD had been altered as a result of mutation for the cumD gene, but any mutation in cumD was not detected in sequence analysis. Comparison of growth of 5 mutants ( selected from 25 mutants ) with that of wild-type IP10 indicates that the activity of a series of enzymes in the cumene degradation pathway, or mechanism of transcriptional control of the cum gene, may have altered in the 5 mutants. Less

异丙苯（异丙苯）是一种芳香烃，其化学结构与甲苯和联苯的化学结构接近。荧光假单胞菌IP01可以在枯烯或甲苯作为唯一碳源和能源上生长，但不能在联苯上生长。我们推测菌株IP 01的生长底物特异性是由于间位裂解化合物水解酶（CumD）的底物特异性，本研究试图通过定点突变的方法确定CumD中负责底物特异性的残基。通过对CumD与其他水解酶氨基酸序列的比较，发现CumD的催化中心有几个保守的氨基酸残基，即Ser^<103>、Asp^<224>、His^<252>，它们的排列顺序与丝氨酸水解酶相似。这三个残基中的每一个被Ala取代使枯烯的间位裂解化合物（HOMODA）水解的酶活性降低到5%以下。的 ...更多信息实验结果支持Ser^<103>、Asp^<224>、His^<252>构成CumD的催化三联体的假设，Trp取代Ile^和Phe<256>取代Trp^<143>可提高间位裂解联苯化合物（HOPDA）的水解活性。这一结果表明Ile^<256>和Trp^<143>是改变CumD底物专一性从而水解HOPDA的关键残基，同时也表明瓦尔<227>^被Ile取代可提高HOMODA的水解活性，这一结果表明瓦尔^在<227>拓扑学上位于催化三联体附近，参与CumD的底物结合位点。在这些突变体中，预测CumD的底物特异性因cumD基因突变而改变，但在序列分析中未检测到cumD中的任何突变。比较5个突变体（从25个突变体中选出）与野生型IP 10的生长表明，枯烯降解途径中的一系列酶的活性或cum基因的转录控制机制可能在5个突变体中发生了改变。少