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

• 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
• 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

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）：天然产品是农业和药物应用的生物活性化合物的极为重要的来源。涉及继发代谢的酶具有巨大的潜力，因为可以在有效合成精细化学物质和高价值药物的生物催化剂中。在代谢工程组与结构生物学组之间的这项合作工作中，我们将收获这一潜力，以一步综合辛伐他汀（Zocor。）。辛伐他汀目前是通过自然产物lovastatin通过效率低下的多步过程合成的。我们提出的辛伐他汀的生物合成将导致一个完全新颖的过程，这可能是当前化学途径的一种有吸引力的替代方法。这项研究中的中心酶是Lovd，这是一种来自洛伐他汀生物合成途径的酰基转移酶。我们已经对这种酶进行了广泛的初步生化特征，以表明LOVD是辛伐他汀合酶，并且可以潜在地设计为辛伐他汀生物合成的强大生物催化剂。该建议将研究以下具体目的： 目标1：Lovd的指向演变。我们将使用定向的进化方法来提高LOVD对辛伐他汀合成的催化效率。我们基于辛伐他汀的形成开发了高吞吐量筛选测定法。 AIM 2：LOVD基于结构的工程。 LOVD的X射线晶体结构将在叶芝实验室中进行。从结构分析中鉴定出的关键残基的合理诱变将进行探测LOVD功能并改善LOVD催化特性，以探测辛伐他汀合成。 AIM 3：大肠杆菌作为整个细胞生物催化剂的代谢工程。我们将设计大肠杆菌的多重转运蛋白系统，以提高其在将辛伐他汀导出到细胞外空间方面的效率。这将在高产品浓度下改善整个细胞反应器的转化。 AIM 4：辛伐他汀的直接生物合成来自A. terreus。我们将在代谢上进行工程A. terreus在lovastatin Biosynsis中被阻断，但在辛伐他汀生物合成中有力。项目叙述 我们提出了生化和结构研究，以研究最近从我们的实验室发现的辛伐他汀合酶。我们将使用蛋白质和代谢工程方法开发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中的一个重要里程碑，将酶应用于合成的化合物，因为尚未报道了辛伐他汀，这在商业上很重要。将天然产物生物合成酶纳入有用的生物催化剂的成功结果可能会导致额外的努力，以检查这类引人入胜的酶从预期的生物催化中。同时，拟议工作的结果将为蛋白质工程，酶结构和功能，大肠杆菌膜转运和曲霉代谢提供重要的科学见解。