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 对限制新功能的进化路线的生物物理约束了解得太少。深入 关于蛋白质进化的生物物理约束的研究一直局限于几个模型系统。 这些研究表明，上位性，当突变在不同的基因组中产生不同的影响时， 序列背景，是常见的，但其生物物理基础和普遍性没有得到很好的研究。滥交， 这是一种偶然的能力，可以进行一种非生物功能的反应， 为自然选择进化新的生物功能提供了原材料。潜在的滥交和 上位性是蛋白质生物物理学：结构，稳定性，动力学和酶机制。这个目标 一个建议是通过比较生物物理学，阐明混杂和上位性在蛋白质进化中的作用。 限制混杂酶的高度特异性酶。我们建立了N-琥珀酰氨基 酸性消旋酶（NSAR）/邻琥珀酰苯甲酸合酶（OSBS）亚家族作为最佳模型之一， 决定了混杂在酶进化中的作用。NSAR活动是从祖先的OSBS进化而来的， 许多酶对这两种活性都是催化混杂的。那些有NSAR活性的也表现出 底物混杂性，优选疏水性N-琥珀酰氨基酸，但与其它侧具有弱活性 店该提案假设滥交与生物物理的性质和程度相关 对进化新活动所需的突变的约束。我们的目标是1）定义生物物理 限制进化的NSAR活性在一个高度特异性的OSBS酶。我们将检验这个假设 几种高度特异性的OSBS由于上位性和其他原因将通过不同的途径进化NSAR活性， 2）比较对混杂NSAR/OSBS酶的生物物理约束， 高度特异性的OSBS酶通过测试假设，改变N-琥珀酰氨基酸的偏好， 混杂的NSAR/OSBS酶的改变将比改变高度NSAR/OSBS酶的底物偏好更可行。 具体的OSBS;和3）制定一个通用的方法来确定上位相互作用。这些目标将是 重要的一步，以确定如何结构，稳定性，动力学和催化机制影响 新酶活性的进化。比较混杂的和高度特异性的酶将阐明 滥交生物物理分析将揭示上位性机制，特别是远处突变的影响 活动现场。我们将利用这些数据，对突变表型进行大规模并行分析， 识别上位相互作用的一般方法。我们将用这种方法来完善蛋白质工程 战略，转向上位性位点，需要同时优化诱变。

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.