Novel Substrate Oxidation by Enzyme Engineering

描述(由申请人提供)：了解P450所显示的各种化学物质是生物医学科学中的一个中心问题，需要了解它们臭名昭著的特异性和混杂倾向。药物代谢的P450在底物识别方面表现出显著的混杂，而参与类固醇生物合成的酶在底物结合和催化方面往往是非常特异的。然而，这些不同形式控制这种混杂或专一性的分子机制还不是很清楚。结构是如何编码这种多样性的？众所周知，在底物识别过程中，许多(如果不是全部)P450发生了显著的构象变化。这些变化也可能在O2激活后的反应门控中发挥作用，以产生活性中间体。然而，我们对底物是如何被特定的P450识别的，或者这些变化是如何耦合到功能上的知之甚少。P450是通过诱导配对还是通过动态采样几个首选构象来识别其底物？过去一段时间的结果对底物与P450结合时发生的构象变化产生了重要的见解，它们表明这些变化可能如何与功能耦合，并为开发具有设计特异性的新型催化剂提供了一个平台。目前的AIMS将使用P450活性部位的分子探针库来控制拟议的活性中间体的形成和性质。根据我们最近的进展，这些研究将测试特定的假设，即活性部位的构象变化如何有助于控制O2激活步骤。我们将开发这些方法来研究特定连接到电极表面的P450的电化学行为。最后，我们将探索使用特定的探针来研究具有设计底物特异性的新型催化剂的分子进化的可能性。这些研究将有助于更好地理解这些重要酶的蛋白质结构是如何与功能和底物专一性相结合的。 与公共卫生相关：P450是一类对人类健康至关重要的各种酶，因为它们负责重要化合物的生物合成和药物代谢。我们的长期目标是开发一种将新的活性引入这些酶的通用方法，因为进化出的P450催化剂有可能生产新的药物和抗生素。在这个过程中，我们将更深入地了解底物是如何识别的，以及酶反应是如何控制的。

DESCRIPTION (provided by applicant): Understanding the diverse chemistry displayed by P450s is a central issue in biomedical science and requires understanding their notorious propensity for specificity and promiscuity. Drug metabolizing P450s display a remarkable promiscuity in substrate recognition, while enzymes involved in steroid biosynthesis are often very specific with respect to substrate binding and catalysis. However, the molecular mechanism by which these different forms control this promiscuity or specificity is not well understood. How does structure encode such diversity? It is known that significant conformational changes occur in many, if not all, P450s during substrate recognition. These changes may also play a role in gating the reactions following O2 activation to produce reactive intermediates. However, we know very little about how substrates are recognized by a particular P450 or how these changes are coupled to function. Does a P450 recognize its substrate by induced fit or by dynamically sampling only a few preferred conformations? Results in the past period have produced significant insights into the conformational changes that occur upon substrate binding to P450, they suggest how these changes may be coupled to function, and they provide a platform for development of novel catalysts with designed specificity. The current aims will use a library of molecular probes for the P450 active site to control the formation and properties of proposed reactive intermediates. These studies will test specific hypotheses, based on our recent progress, about how conformational changes at the active site may help control the O2 activation step. We will develop these approaches to investigate the electrochemical behavior of P450s specifically wired to electrode surfaces. Finally, we will explore the potential for using specific probes for molecular evolution of novel catalysts with designed substrate specificity. These studies will contribute to a better understanding how the protein structure of these important enzymes is coupled to function and substrate specificity. PUBLIC HEALTH RELEVANCE: P450s are a diverse class of enzymes of critical importance to human health because they are responsible for the biosynthesis of important compounds and drug metabolism. Our long term goal is to develop a general method for introducing new activities into these enzymes, as evolved P450 catalysts have the potential for producing novel drugs and antibiotics. In the process, we will gain a deeper understanding of how the substrates are recognized and how the enzymatic reactions are controlled.