Biochemical and Structural Studies of A Novel Simvastatin Synthase

描述（由申请人提供）：天然产品是农业和制药应用的生物活性化合物的极其重要的来源。参与次生代谢的酶作为生物催化剂在精细化学品和高价值药物的高效合成中具有巨大的潜力。在代谢工程小组和结构生物学小组之间的这项合作中，我们将收获一步合成重磅药物辛伐他汀（Zocor.）的潜力。辛伐他汀目前是由天然产物洛伐他汀通过低效的多步工艺合成的。我们提出的辛伐他汀的生物合成将导致一个全新的过程，可以是一个有吸引力的替代目前的化学路线。本研究中的中心酶是LovD，一种来自洛伐他汀生物合成途径的酰基转移酶。我们已经进行了广泛的，初步的生化特性，这种酶表明，LovD是辛伐他汀合成酶，并可能被改造成一个强大的生物催化剂辛伐他汀生物合成。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。我们已经开发了一个高通量筛选分析的基础上形成的辛伐他汀。 目的2：LovD的基于结构的工程。LovD的X射线晶体结构将在耶茨实验室进行。将对从结构分析中鉴定的关键残基进行合理诱变，以探测LovD功能并改善LovD对辛伐他汀合成的催化性质。 目的3：E.大肠杆菌作为全细胞生物催化剂。我们将设计E.大肠杆菌，以提高其输出辛伐他汀到细胞外空间的效率。这将提高高产物浓度下全电池反应器的转化率。 目的4：从A. terreus我们将代谢工程A。terreus在洛伐他汀生物合成中被阻断，但在辛伐他汀生物合成中是稳健的。项目叙述 我们已经提出了生物化学和结构的研究，调查辛伐他汀合酶最近确定从我们的实验室。我们将利用蛋白质和代谢工程的方法，开发一个全细胞生物催化剂，可以生物合成辛伐他汀。这项工作将代表生物催化的一个重要里程碑，酶对合成的化合物，如辛伐他汀的商业重要的应用还没有报道。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从生物催化的前景来检查这类迷人的酶的额外努力。同时，这项工作的结果将为蛋白质工程、酶的结构和功能、大肠杆菌的研究提供重要的科学见解。大肠杆菌膜转运和曲霉代谢。

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.