ENGINEERED SUBSTRATE SPECIFICITIES OF CYTOCHROME P450S

细胞色素P450酶是主要的酶， 氧化和还原代谢的许多内源性和 异生物质基质 P450催化多种 反应的类型及其广泛的底物特异性使得这些 酶是一个很好的选择，工程努力，创造新的 底物特异性 我们希望设计CYP119，第一个P450 迄今为止，只有b型血红素蛋白被鉴定出来， 嗜酸、嗜热古细菌转化为自给自足的P450 能够代谢非天然底物。 CYP119是 最初克隆自 硫磺硫化叶菌（Sulfolobus solfataricus），一种需氧、嗜酸热古菌， 基于其与其他P450的序列同源性， P450超家族成员 由于其嗜酸性和嗜热性，CYP 119是一种 比目前可用的模型系统更通用的模型系统。 dock是 这是一个重要的工具，有助于我们完善对P450的理解， 底物特异性 DOCK已经用野生型P450cam进行了测试 和两个变种人 更广泛的P450的进一步研究 和潜在的基板需要作出的办法， 实用性 CYP119的同源性模型正在构建中 基于四个结晶P450的比对 重要 使用Midas鉴定活性位点中的残基。 将使用定点诱变来评估它们在以下方面的作用： 蛋白质的特异性和活性。 我们希望结果 来自P450 cam、P450 terp、P450 eryf、P450 BM-3和CYP 119研究 将为正确预测提供必要的信息。 这些突变将影响催化剂中所需的变化， P450的特异性

Cytochrome P450s are the main enzymes responsible for the oxidative and reductive metabolism of numerous endogenous and xenobiotic substrates. The ability of the P450s to catalyze multiple types of reactions and their broad substrate specificity make these enzymes an excellent choice for engineering efforts to create new substrate specificities. We hope to engineer CYP119, the first P450 and only b-type hemeprotein to be identified thus far from any acidophilic, thermophilic archaebacteria, into a self-sufficient P450 capable of metabolizing non- natural substrates. CYP119 was originally cloned from Sulfolobus solfataricus, an aerobic, acidothermophilic archaeon, and identified as a potential P450 based n its sequence homology to other members of the P450 superfamily. Because of its acidophilic and thermophilic properties, CYP119 is a more versatile model system than those currently available. DOCK is an important tool that is helping to refine our understanding of P450 substrate specificity. DOCK has been tested with wild-type P450cam and two of its mutants. Further studies with a wider range of P450s and potential substrates are required to make the approach of practical utility. A homology model of CYP119 is being constructed based on alignments with the four crystallized P450s. Important residues in the active site will be identified using Midas. Site-directed mutagenesis will be used to assess their role in specificity and activity of the protein. We hope that the results from studies with P450cam, P450terp, P450eryf, P450BM-3 and CYP119 will provide the necessary information for the correct prediction of mutations that will effect desired changes in the catalytic specificity of P450s.