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。从天然来源中分离得到的酶因其选择性好、催化反应种类多、反应条件温和、反应速度快等特点，在化工、生物技术和医疗保健等领域得到了广泛的应用。然而，不利的内在性质的酶，包括边际稳定性，狭窄的底物特异性和不兼容的非水溶剂，使工程酶的必要。对于某些应用，酶可以重新设计以催化自然界中未发现的反应。因此，我们设计和重新设计高效酶的能力具有极其重要的实际意义。 对酶进行合理的重新设计，以提高催化活性和稳定性，是对我们理解蛋白质序列-结构-功能关系的最终考验。虽然计算工具的进步已经能够构建催化非自然反应的新酶，但我们以合理的方式大幅提高酶活性以实现所需反应的能力仍然不发达。相比之下，定向进化实验通过随机突变和选择来提高适应度，是一种非常成功的改善酶功能的方法。然而，我们对有益的随机突变和适应性景观的结构和机制基础的理解仍然是初步的。因此，全面研究大的序列变化如何导致酶功能的巨大变化，不仅可以弥合蛋白质序列-结构-功能关系的基本知识差距，还可以显着提高我们设计具有所需特性的定制酶的能力。 该方案试图通过结合蛋白质工程实验室（Yi Tang）、结构生物学实验室（托德耶茨）和计算蛋白质设计实验室（Ken Houk）的专业知识来揭示蛋白质适应度景观的结构和机制基础。四个相互关联和跨学科的目标将探索最近发现的酶LovD的催化景观，LovD的活性在实验室中朝着合成降胆固醇药物辛伐他汀的方向新发展：1）LovD酶进行新反应的定向进化途径中的突变体的结构分析; 2）LovD和突变体的生物化学和生物物理研究; 3）LovD突变体的计算表征;和4）预期赋予LovD增强的催化活性的替代序列突变的计算预测。