Biochemical and Structural Studies of A Novel Simvastatin Synthase

新型辛伐他汀合酶的生化和结构研究

基本信息

批准号：
7355623
负责人：
Yi Tang
金额：
$ 18.44万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7355623
关键词：
Active Sites Acyltransferase Address Agriculture Anabolism Aspergillus Binding Biochemical Biological Assay Biological Factors Biomedical Engineering Cells Chemicals Cholesterol Class Complex Engineering Enzymes Escherichia coli Extracellular Space Goals Harvest Laboratories Lead Lovastatin Metabolic Metabolism Methods Molecular Conformation Mutagenesis Natural Product Drug One-Step dentin bonding system Outcome Pathway interactions Pharmaceutical Preparations Pharmacologic Substance Process Production Property Protein Engineering Protein Overexpression Proteins Reporting Roentgen Rays Route Sales Simvastatin Site-Directed Mutagenesis Source Structure System Time Transmembrane Transport Work base chemical reaction chemical synthesis cost directed evolution enzyme structure fascinate high throughput screening improved improved functioning in vivo insight microorganism multi drug transporter novel prospective structural biology thioester zocor

项目摘要

DESCRIPTION (provided by applicant): Natural products are extremely important sources of bioactive compounds for agricultural and pharmaceutical applications. Enzymes involved in secondary metabolism hold great potential as biocatalysts that may be used in the efficient synthesis of fine chemicals and high value pharmaceuticals. In this collaborative work between a metabolic engineering group and a structural biology group, we will harvest this potential towards the one-step synthesis of the blockbuster drug simvastatin (Zocor.). Simvastatin is currently synthesized from the natural product lovastatin via inefficient, multistep processes. Our proposed biosynthesis of simvastatin will result in a completely novel process that can be an attractive alternative over the current chemical routes. The central enzyme in this study is LovD, an acyltransferase from the lovastatin biosynthetic pathway. We have performed extensive, preliminary biochemical characterization of this enzyme to show that LovD is a simvastatin synthase, and can be potentially engineered into a powerful biocatalyst for simvastatin biosynthesis. This proposal will examine the following specific aims: AIM 1: Directed Evolution of LovD. We will use directed evolution methods to improve the catalytic efficiencies of LovD towards simvastatin synthesis. We have developed a high throughput screening assay based on the formation of simvastatin. AIM 2: Structure-Based Engineering of LovD. The X-ray crystal structure of LovD will be pursued in the Yeates Lab. Rational mutagenesis of key residues identified from structural analysis will be performed to probe LovD function and improve LovD catalytic properties towards simvastatin synthesis. AIM 3: Metabolic Engineering of E. coli as a whole cell biocatalyst. We will engineer the multidrug transporter system of E. coli to improve its efficiency in exporting simvastatin to the extracellular space. This will improve the conversion of the whole cell reactor at high product concentrations. AIM 4: Direct Biosynthesis of Simvastatin from A. terreus. We will metabolically engineer A. terreus to be blocked in lovastatin biosynthesis, but robust in simvastatin biosynthesis. Project Narrative We have proposed biochemical and structural studies to investigate a simvastatin synthase recently identified from our laboratories. We will use protein and metabolic engineering methods to develop a whole cell biocatalyst that can biosynthesize simvastatin. This work will represent an important milestone in biocatalysis, application of enzymes towards the synthesis of a compound as commercially important as simvastatin has not been reported. The successful outcome of engineering a natural product biosynthetic enzyme into a useful biocatalyst may lead to additional efforts to examine this class of fascinating enzymes from a biocatalysis prospective. At the same time, the outcome of the proposed work will provide important scientific insight into protein engineering, enzyme structure and function, E. coli membrane transport, and Aspergillus metabolism.

描述（由申请人提供）：天然产物是农业和制药应用中极其重要的生物活性化合物来源。参与二次代谢的酶作为生物催化剂具有巨大的潜力，可用于精细化学品和高价值药物的有效合成。在代谢工程小组和结构生物学小组之间的这项合作工作中，我们将收获一步合成重磅药物辛伐他汀（Zocor.）的潜力。辛伐他汀目前是通过低效的多步骤工艺从天然产物洛伐他汀合成的。我们提出的辛伐他汀生物合成将产生一种全新的工艺，该工艺可以成为当前化学路线的有吸引力的替代方案。本研究的中心酶是 LovD，一种来自洛伐他汀生物合成途径的酰基转移酶。我们对该酶进行了广泛的初步生化表征，表明 LovD 是一种辛伐他汀合酶，并且有可能被设计成用于辛伐他汀生物合成的强大生物催化剂。该提案将审查以下具体目标：目标 1：LovD 的定向进化。我们将使用定向进化方法来提高LovD对辛伐他汀合成的催化效率。我们开发了一种基于辛伐他汀形成的高通量筛选测定法。目标 2：LovD 基于结构的工程。 Yeates 实验室将研究 LovD 的 X 射线晶体结构。对结构分析确定的关键残基进行合理诱变，以探测 LovD 功能并提高 LovD 对辛伐他汀合成的催化性能。目标 3：大肠杆菌作为全细胞生物催化剂的代谢工程。我们将设计大肠杆菌的多药物转运系统，以提高其将辛伐他汀输出到细胞外空间的效率。这将提高整个电池反应器在高产物浓度下的转化率。目标 4：从土曲霉中直接生物合成辛伐他汀。我们将通过代谢改造土曲霉，使其在洛伐他汀生物合成中被阻断，但在辛伐他汀生物合成中保持稳健。项目叙述我们提出了生物化学和结构研究来研究我们实验室最近鉴定的辛伐他汀合酶。我们将利用蛋白质和代谢工程方法开发一种可以生物合成辛伐他汀的全细胞生物催化剂。这项工作将代表生物催化领域的一个重要里程碑，应用酶来合成像辛伐他汀这样具有商业重要性的化合物尚未有报道。将天然产物生物合成酶工程化为有用的生物催化剂的成功结果可能会导致人们进一步努力从生物催化的角度研究这类令人着迷的酶。同时，拟议工作的成果将为蛋白质工程、酶结构和功能、大肠杆菌膜运输和曲霉代谢提供重要的科学见解。