Interconversion of Specificity within Enzyme Families

描述（由申请人提供）：了解酶超家族成员的功能和底物特异性如何多样化是蛋白质化学中的一个关键问题，对进化、蛋白质设计和生物医学应用中有用酶的工程化具有深远意义。在这里，我们提出了一个全面的和高度集成的实验研究计划，探索如何相互转换的酶超家族成员之间的底物特异性表现出低程度的氨基酸序列同一性。作为这项工作的一部分，全新的嵌合酶，来自组合组装的亚结构域从亲本序列，将被分离出高度多样化的图书馆。酶分离和文库功能和底物特异性的询问将通过主要利用单细胞流式细胞术测定的定量、超高通量筛选来完成。嵌合酶表现出所需的催化活性和底物选择性的配置文件将被结晶，高分辨率的结构将获得在可能的情况下，最后，酶的催化机制将被详细分析。作为这项研究的一部分，我们将研究如何组合组装的蛋白质亚结构域可以采用相互转换的丝氨酸蛋白酶（弹性蛋白酶和糜蛋白酶）的特异性和转换的特异性的人谷胱甘肽S转移酶的大鼠酶。与此同时，我们将探索由选择用于组合的两个亲本基因的氨基酸序列同一性降低所施加的限制，并寻求通过诱变来克服这些限制。基因对将从二氢叶酸还原酶序列的广泛家族中选择，允许42%至28%序列同一性的系统变异-在所有情况下都低于经典DNA改组。因此，拟议的研究将有助于描绘二级结构元件和特定的氨基酸，决定：（a）在胰蛋白酶的切割特异性;（B）GST酶的谷胱甘肽共轭亲电底物的识别，最后（c）蛋白质折叠和二氢叶酸还原酶的催化活性。还将研究具有不同于任一亲本的新型底物特异性谱的酶的产生。最后，但也许同样重要的是，这项工作将验证一个独特的，高度跨学科的方法，探索和更深入地了解酶的功能。

DESCRIPTION (provided by applicant): Understanding how function and substrate specificity are diversified within the members of an enzyme superfamily represents a key question in protein chemistry with profound implications for evolution, protein design and for the engineering of useful enzymes for biomedical applications. Here we present a comprehensive and highly integrated experimental research program for exploring how to interconvert substrate specificity among enzyme superfamily members exhibiting a low degree of amino acid sequence identity. As part of this work, entirely new chimeric enzymes, derived from the combinatorial assembly of subdomains from parental sequences, will be isolated from highly diverse libraries. Enzyme isolation and the interrogation of the libraries for function and substrate specificity will be accomplished by virtue of quantitative, ultra-high throughput screening that capitalizes predominantly on single cell, flow cytometric assays. Chimeric enzymes exhibiting desired profiles of catalytic activity and substrate selectivity will be crystallized, high resolution structures will be obtained where possible and finally, the catalytic mechanism of the enzymes will be analyzed in detail. As part of this study we will examine how the combinatorial assembly of protein subdomains can be employed to interconvert the specificity of serine proteases (elastase and chymotrypsin) and to transform the specificity of the human glutathione S transferase to that of the rat enzyme. In parallel we will explore the limits imposed by the decreasing amino acid sequence identity for the two parental genes selected for combination and seek to overcome these limits by mutagenesis. The gene pairs will be chosen from the extensive family of dihydrofolate reductase sequences allowing a systematic variation from 42% to 28% sequence identity--in all cases below that of classical DNA shuffling. Consequently, the proposed studies will help delineate the secondary structural elements and specific amino acids that dictate: (a) the cleavage specificity in trypsin proteases; (b) recognition of electrophile substrates in glutathione conjugation by GST enzymes and finally (c) protein folding and catalytic activity in dihydrofolate reductase. The generation of enzymes having novel substrate specificity profiles distinct from either parent will also be investigated. Finally, but perhaps equally importantly, this work will validate a unique, highly interdisciplinary approach for the exploration and deeper understanding of enzyme function.