ENGINEERING SUBSTRATE SPECIFICITIES OF CYTOCHROMES P450

细胞色素P450是负责细胞凋亡的主要酶。 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 我们希望工程CYP 119，第一个 到目前为止，嗜热P450被鉴定为自给自足的 P450能够代谢非天然底物。 CYP 119是 最初克隆自硫磺硫化叶菌，一种需氧， 嗜酸嗜热古菌，并确定为一个潜在的P450为基础的 与P450超家族其他成员的序列同源性。 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。 一 CYP 119的同源性模型已经基于与 四个结晶的P450 使用MidasPlus，重要的活动站点 已鉴定并突变了残基。 T213的初步研究 突变体表明该残基在特异性 和CYP 119的活性。 此外，我们正在准备运行MD 对野生型和突变型CYP 119进行模拟，以研究 温度的变化。 DOCK也是一个重要的 这是一个帮助我们更好地理解P450底物的工具。 的特异性 DOCK已经用野生型P450 cam和两种 它的突变体。 进一步研究更广泛的P450， 仍然需要潜在的基底来实现这种方法， 实用性 我们希望研究的综合结果 与P450 cam，P450 terp，P450 eryf，P450 BM-3和CYP 119将提供 正确预测突变的必要信息， 将影响P450的催化特异性的所需变化。

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 thermophilic P450 to be identified thus far, 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 on 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. A homology model of CYP119 has been constructed based on alignments with the four crystallized P450s. Using MidasPlus, important active site residues have been identified and mutated. Initial studies with T213 mutants indicate a prominent role for this residue in the specificity and the activity of CYP119. In addition, we are preparing to run MD simulations on wild-type and mutant CYP119 to investigate the impact of temperature on active site topology. DOCK is also 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 still required to make this approach of practical utility. We hope that the combined 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.