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

异丙苯（异丙苯）是一种芳香烃，其化学结构与甲苯和联苯相似。P.fluorescens IP01可以在异丙烯或甲苯作为唯一的碳源和能源上生长，但不能在联苯上生长。我们推测菌株IP01的生长底物特异性归因于元裂解复合水解酶（CumD）的底物特异性。在这项研究中，我们试图通过位点定向诱变确定在CumD中负责底物特异性的残基。通过比较CumD与其他水解酶的氨基酸序列，我们发现了几个可能在CumD催化中心起功能作用的保守残基，即Ser^<103>, Asp^<224>, His^<252>这三个氨基酸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，使异丙茴香（homda）间裂解化合物水解的酶活性降低到5%以下。结果支持了Ser^<103>、Asp^<224>、His^<252>构成CumD催化三元组的假设。色氨酸取代Ile^<256>和苯丙氨酸取代Trp^<143>增加了联苯（HOPDA）间裂解化合物水解的酶活性。这表明Ile^<256>和Trp^<143>是改变CumD底物特异性从而使HOPDA水解的关键残基。结果还表明，用Ile取代Val^<227>可提高homda水解酶的活性。这一结果表明，Val^<227>在拓扑结构上位于催化三联体附近，并参与了CumD的底物结合位点。IP01在含有少量异丙烯的联苯上连续培养，获得了25个高效降解联苯的突变体。在这些突变体中，预测CumD的底物特异性由于CumD基因的突变而改变，但在序列分析中未检测到任何CumD突变。从25个突变体中选择的5个突变体与野生型IP10的生长比较表明，这5个突变体可能改变了cumene降解途径中一系列酶的活性，或改变了cumene基因的转录控制机制。少

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