Mapping the Evolution of a Novel Enzyme by Experiment and Computation

通过实验和计算绘制新型酶的进化图

• 批准号: 8625310
• 负责人: KENDALL N HOUK
• 金额: $ 33.83万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8625310
• 关键词: Active Sites; Affinity; Amino Acid Sequence; Amino Acids; Anabolism; Binding; Biochemical; Biological Sciences; Biotechnology; Catalysis; Chemicals; Cholesterol; Computing Methodologies; Custom; DNA Sequence Rearrangement; Data; Engineering; Entropy; Enzyme Stability; Enzymes; Evolution; Free Energy; Gene Mutation; Geometry; Goals; Healthcare Industry; Investigation; Kinetics; Knowledge; Laboratories; Lead; Maps; Methods; Modeling; Mutagenesis; Mutation; Nature; Pathway interactions; Peptide Sequence Determination; Pharmaceutical Preparations; Property; Protein Conformation; Protein Engineering; Proteins; Reaction; Role; Series; Side; Simvastatin; Solvents; Source; Structure; Structure-Activity Relationship; Substrate Specificity; Testing; Triad Acrylic Resin; Water; base; biophysical properties; catalyst; computerized tools; design; directed evolution; enzyme activity; fitness; improved; insight; mutant; novel; protein protein interaction; reaction rate; research study; simulation; structural biology; tool

DESCRIPTION (provided by applicant): Enzymes are the most versatile catalysts. Because of their exquisite selectivity, diverse array of catalyzed reactions, mild reaction conditions, and significant enhancement of reactions rates, enzymes isolated from natural sources have been widely used in the chemical, biotechnology and health care industries. However, unfavorable intrinsic properties of enzymes, including marginal stability, narrow substrate specificity and incompatibility with nonaqueous solvents, have made engineering of enzymes necessary. For some applications, enzymes can be designed de novo to catalyze reactions that are not found in nature. Therefore, our abilities to design and redesign efficient enzymes have extremely important and practical implications. The rational redesign of enzymes towards increased catalytic activity and stability is an ultimate test of our understanding of protein sequence-structure-function relationships. Although advances in computational tools have enabled construction of new enzymes catalyzing unnatural reactions, our ability to drastically improve enzyme activity towards a desired reaction in a rational manner has remained underdeveloped. In contrast, directed evolution experiments, in which fitness is elevated via random mutation and selection, is a highly successful method of improving enzyme function. However, our understanding of the structural and mechanistic basis of beneficial random mutations and fitness landscape remains rudimentary. Therefore, a comprehensive investigation of how large sequence changes can lead to dramatic changes in enzyme function will not only bridge this fundamental knowledge gap in protein sequence-structure-function relationships, it will also significantly improve our capabilities in designing custom enzymes with desired properties. This proposal attempts to reveal the structural and mechanistic bases of protein fitness landscape by combining the expertise of a protein engineering lab (Yi Tang), a structural biology lab (Todd Yeates) and a computational protein design lab (Ken Houk). Four interrelated and interdisciplinary aims will explore the catalytic landscape of a recently discovered enzyme, LovD, whose activity has been newly evolved in the laboratory towards the synthesis of the cholesterol lowering drug simvastatin: 1) Structural analysis of mutants in the directed- evolutionary pathway of a LovD enzyme carrying out a new reaction; 2) Biochemical and biophysical studies of LovD and mutants; 3) Computational characterization of LovD mutants; and 4) Computational prediction of alternate sequence mutations expected to confer enhanced catalytic activity on LovD.

描述（由申请人提供）：酶是最通用的催化剂。由于其精致的选择性，各种各样的催化反应，轻度的反应条件以及反应速率的显着提高，从天然来源中分离出来的酶已被广泛用于化学，生物技术和保健行业。然而，酶的不利固有特性，包括边际稳定性，狭窄的底物特异性和与非水溶剂的不相容性，使得必要的酶工程化。对于某些应用，可以从头设计酶来催化自然界未发现的反应。因此，我们设计和重新设计有效酶的能力具有极其重要和实际的含义。 酶对增加催化活性和稳定性的有理重新设计是我们对蛋白质序列结构功能关系的理解的最终检验。尽管计算工具的进步已经促进了催化非自然反应的新酶的构建，但我们以合理方式将酶活性大幅提高酶活性的能力仍然欠发达。相反，通过随机突变和选择来提高适应性的定向进化实验是改善酶功能的一种非常成功的方法。但是，我们对有益随机突变和健身景观的结构和机械基础的理解仍然是基本的。因此，对较大序列变化如何导致酶功能的巨大变化的全面研究不仅会弥合蛋白质序列结构 - 功能关系中的这一基本知识差距，而且还将显着提高我们在设计具有所需特性的自定义酶方面的能力。 该建议试图通过结合蛋白质工程实验室（YI Tang），结构生物学实验室（TODD Yeates）和计算蛋白设计实验室（KEN HOUK）的专业知识来揭示蛋白质健身景观的结构和机械基础。四个相互关联和跨学科的目的将探索最近发现的酶，Lovd的催化景观，该酶的活性在实验室中新进化，用于合成降低胆固醇的药物辛伐他汀的合成：1）突变体在直接进化途径中的结构性分析一个新反应的Lovd酶； 2）LOVD和突变体的生化和生物物理研究； 3）LOVD突变体的计算表征； 4）预期在LOVD上赋予增强催化活性的替代序列突变的计算预测。