Novel Substrate Oxidation by Enzyme Engineering

描述(由申请人提供)：在这次更新中，我们进入了我们长期目标的第三阶段，以了解血红素酶的各种反应活性，并利用这些信息来产生具有新催化特性的工程形式。推动这些研究的主要假设是，血红素酶表现出的化学反应活性部分但不完全位于血红素辅助因子内，蛋白质的一个重要作用是限制或直接底物以导致特定催化的方式进入这个反应中心。显然，这一假设是不成立的，因为存在许多蛋白质直接调节血红素活性的例子。因此，我们的目标是划定蛋白质这两个角色之间的边界，然后利用这些信息引入新的底物与血红素辅助因子相互作用的新位点，利用现有的血红素-蛋白质相互作用，并诱导新的催化反应。该项目的第一阶段的特点是强调血红素酶的物理、光谱和功能特性，主要是以细胞色素c过氧化物酶(CCP)为模型。这项工作支持了一些统一的主题，这些主题表明一些血红素酶的反应可能会被招募到其他酶的支架上。在第二阶段，我们开发了一种方法，通过“空位互补”将小分子结合位点引入到血红素环境中，即通过突变删除氨基酸侧链来创建结合位点。这一方法为测试有关血红素酶功能多样性的想法提供了新的方法。它还遇到了Rational工程的一些限制，无法生成具有定制功能的酶。在下一阶段，我们寻求开始开发一种实验方法，用于结合研究人员选择的化合物的血红素蛋白质和酶的定向进化。进化的蛋白质将基于细菌细胞色素P450和亚硝基，并将利用噬菌体展示来选择将目标化合物结合在相对于血红素的特定位置的突变体。我们的总体目标是确定这些不同蛋白质支架的配体结合位点是否可以被诱导以预定的方式结合各种靶标配体。如果是这样，那么设计具有特定特异性的新型催化剂的通用方法可能是可能的。此外，关于特定蛋白质结构接受给定配体的固有倾向的信息应该提供对P450底物特异性的基本原理的洞察。

DESCRIPTION (provided by applicant): In this renewal, we enter a third phase of our long term goal to understand the diverse reactivity of heme enzymes and to use that information to generate engineered forms with novel catalytic properties. The primary hypothesis that has driven these studies is that the chemical reactivity displayed by heme enzymes resides partially, but not exclusively, within the heme cofactor, and a significant role of the protein is to limit or direct access of substrates to this reactive center in ways that result in specific catalysis. Clearly, this hypothesis falls short, for many examples exist where the protein directly modulates the activity of the heme. It has thus been our goal to delineate the boundaries between these two roles for the protein, and then use this information to introduce new sites where novel substrates interact with the heme cofactor, take advantage of existing heme-protein interactions, and induce novel catalytic reactions. The first phase of this project was characterized by its emphasis on the physical, spectroscopic and functional properties of heme enzymes, primarily cytochrome c peroxidase (CCP) as a model. This work has given support to unifying themes that suggest how reactions of some heme enzymes might be recruited into the scaffold of others. In the second phase, we have developed an approach to introduce small-molecule binding sites into the heme environment by "cavity complementation", the creation of binding sites by mutagenic deletion of amino acid side-chains. This approach has provided new ways to test ideas about the diversity of heme enzyme function. It has also come up against some of the limitations of rational engineering to generate enzymes with tailor-made functionality. In the next period, we seek to begin development of an experimental method for the directed evolution of heme proteins and enzymes that bind compounds of the investigator's choice. The evolved proteins will be based upon bacterial cytochrome P450s and nitrophorins, and will utilize phage display to select mutants that bind the target compound in a defined position with respect to the heme. Our overall goal is to determine if the ligand binding sites of these different protein scaffolds can be induced to bind a variety of target ligands in a predetermined way. If so, then a general method for engineering novel catalysts with defined specificity may be possible. In addition, information about the inherent propensity of a particular protein architecture to accept a given ligand should provide insight into fundamentals of P450 substrate specificity.