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是如何多样化的，这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具有深远的影响。在这里，我们提出了一个全面和高度整合的实验研究计划，探索如何在表现出低程度氨基酸序列同一性的酶超家族成员之间相互转换底物特异性。作为这项工作的一部分，将从高度多样化的文库中分离出全新的嵌合酶，这些酶来自亲本序列的亚结构域的组合组装。酶的分离和文库的功能和底物特异性的询问将通过定量、超高通量筛选来完成，这种筛选主要利用单细胞、流式细胞术分析。嵌合酶具有理想的催化活性和底物选择性，将在可能的情况下获得高分辨率的结构，最后，将详细分析酶的催化机制。作为本研究的一部分，我们将研究如何利用蛋白质亚结构域的组合组装来相互转换丝氨酸蛋白酶（弹性蛋白酶和凝乳胰蛋白酶）的特异性，并将人类谷胱甘肽S转移酶的特异性转化为大鼠酶的特异性。同时，我们将探讨由选择组合的两个亲本基因的氨基酸序列同一性减少所施加的限制，并寻求通过诱变来克服这些限制。这些基因对将从广泛的二氢叶酸还原酶序列家族中选择，允许系统变异从42%到28%的序列同一性-在所有情况下都低于经典的DNA洗选。因此，拟议的研究将有助于描述二级结构元件和特定氨基酸，这些结构元件和氨基酸决定：(a)胰蛋白酶的裂解特异性；(b) GST酶对谷胱甘肽偶联中亲电底物的识别，最后(c)二氢叶酸还原酶的蛋白质折叠和催化活性。产生具有不同于任何亲本的新型底物特异性谱的酶也将被研究。最后，但也许同样重要的是，这项工作将验证一种独特的、高度跨学科的方法，用于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.