Biophysical constraints on evolution of enzyme specificity

项目摘要 设计与天然酶一样有效的酶是非常困难的，这表明我们 对限制新功能进化路线的生物物理限制因素了解太少。深入探讨 关于蛋白质进化的生物物理约束的研究一直局限于少数几个模型系统。 这些研究表明，上位性，即当突变对不同的 序列背景是常见的，但其生物物理基础和渗透性还没有得到很好的研究。乱交， 这是一种巧合的执行非生物功能的反应的能力，也通过 为自然选择进化新的生物功能提供原材料。潜在的滥交和 上位性是蛋白质的生物物理学：结构、稳定性、动力学和酶机制。这样做的目的是 建议通过比较生物物理来阐明混杂和上位性在蛋白质进化中的作用。 对杂交酶的限制是对高度专一性酶的限制。我们建立了N-琥珀酰氨基 酸性消旋酶(NSAR)/邻丁二酸苯甲酸合成酶(OSBS)亚家族作为最好的模型之一 确定混杂在酶进化中的作用。NSAR活动是从祖先的OSB进化而来的，并且 许多酶对这两种活性都是催化混杂的。那些有NSAR活动的也展示了 底物杂乱，偏爱疏水性N-琥珀酸基氨基酸，但与另一侧活性较弱 锁链。这一建议假设性乱与生物物理的性质和程度有关。 对进化新活动所需的突变的限制。我们的目标是1)定义生物物理学 一种高度特异的OSBS酶对NSAR活性进化的限制。我们将检验这一假设 由于上位性和其他原因，几个高度特异的OSB将通过不同的途径进化NSAR活性 生物物理约束；2)比较混杂NSAR/OSBS酶的生物物理约束和 通过检验改变N-琥珀酸基氨基酸偏好的假说获得高度特异性的OSBS酶 混合NSAR/OSBS酶的选择将比改变高度的底物选择性更可行。 具体的OSB；以及3)开发一种识别上位性交互作用的一般方法。这些目标将成为 朝着确定结构、稳定性、动力学和催化机理如何影响 新的酶活性的进化。比较混杂的和高度特异的酶将澄清 乱交。生物物理分析将揭示上位性机制，特别是远距离突变的影响 活动站点。我们将利用这些数据对突变表型进行大规模并行分析，以开发出 确定上位性交互作用的一般方法。我们将使用这种方法来提炼蛋白质工程 策略，引导突变朝着需要同时优化的上位位置进行。

Project Summary Designing enzymes that are as efficient as natural enzymes is very difficult, showing that we understand too little about the biophysical constraints that limit evolutionary routes to new functions. In-depth studies about the biophysical constraints on protein evolution have been limited to a few model systems. These studies indicate that epistasis, which occurs when mutations have different effects in different sequence contexts, is common, but its biophysical basis and pervasiveness are not well studied. Promiscuity, which is the coincidental ability to carry out a reaction that is not a biological function, also plays a role by providing the raw material for natural selection to evolve new biological functions. Underlying promiscuity and epistasis is protein biophysics: structure, stability, dynamics, and enzymatic mechanism. The goal of this proposal is to illuminate the roles of promiscuity and epistasis in protein evolution by comparing biophysical constraints on promiscuous enzymes to those of highly specific enzymes. We established the N-succinylamino acid racemase (NSAR)/o-succinylbenzoate synthase (OSBS) subfamily as one of the best models for determining the role of promiscuity in enzyme evolution. NSAR activity evolved from an ancestral OSBS, and many enzymes are catalytically promiscuous for both activities. Those that have NSAR activity also exhibit substrate promiscuity, preferring hydrophobic N-succinylamino acids but having weak activity with other side chains. This proposal hypothesizes that promiscuity is correlated with the nature and extent of biophysical constraints on mutations that are required to evolve new activities. Our aims are to 1) Define the biophysical constraints on evolution of NSAR activity in a highly specific OSBS enzyme. We will test the hypothesis that several highly specific OSBSs will evolve NSAR activity by different routes due to epistasis and other biophysical constraints; 2) Compare biophysical constraints on promiscuous NSAR/OSBS enzymes and highly specific OSBS enzymes by testing the hypothesis that changing the N-succinylamino acid preference of promiscuous NSAR/OSBS enzymes will be more feasible than changing substrate preference of highly specific OSBSs; and 3) Develop a general approach to identify epistatic interactions. These aims will be a significant step toward determining how structure, stability, dynamics and catalytic mechanism affect the evolution of new enzyme activities. Comparing promiscuous and highly specific enzymes will clarify the role of promiscuity. Biophysical analysis will reveal epistatic mechanisms, especially effects of mutations distant from the active site. We will leverage this data with a massively parallel analysis of mutation phenotypes to develop a general approach to identify epistatic interactions. We will use this approach to refine protein engineering strategies, steering mutagenesis toward epistatic sites that need to be simultaneously optimized.