Engineering Enzymes for New Stereoselective and Stereodynamic Processes: An Integrated Chemistry -Bioengineering- X-Ray Crystallography-Molecular Dynamics Approach

用于新立体选择性和立体动力学过程的工程酶：化学-生物工程-X射线晶体学-分子动力学综合方法

• 批准号: 2023250
• 负责人: Mark Wilson
• 金额: $ 60.39万
• 依托单位: University of Nebraska-Lincoln
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2025-04-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2023250&HistoricalAwards=false
• 关键词: Engineering; Enzymes; New; Stereoselective; Stereodynamic

Most pharmaceutical drugs and agrochemicals are what is referred to as fine chemicals. The molecules have very specific structures that give them their beneficial properties. Enzymes are highly effective in producing such structures. The objective of this project is to develop a strategy to design and tune the activity of enzymes that are involved in the production of a class of fine chemicals called taxanes. These compounds have anti-cancer properties. One of the taxanes, taxol, is currently used in the treatment of metastatic breast cancers. The project will include educating and mentoring undergraduate and graduate students at the interface of biomolecular engineering, chemistry and structural biology. A collaboration with the science department at a large public high school with a diverse student population is also planned. These efforts will develop a workforce to support a vibrant bioeconomy.The tools of molecular dynamics simulation and x-ray crystallography will guide the directed evolution of two promising nicotinamide-dependent oxidoreductases. The focus is on engineering these enzymes for reactions in which racemic substrates can be effectively deracemized and converted to primarily one of four possible stereoisomers through dynamic reductive kinetic resolution. The targeted transformations will provide access to a spectrum of valuable building blocks for natural products synthesis, chemical biology and medicinal chemistry. Several building blocks to be constructed are for chemotherapeutic agents of the taxane family, useful as anti-cancer drugs. In previous work, the team has shown that the Clostridial enzyme (CaADH) shows remarkable substrate promiscuity, while at the same time exhibits considerable stereochemical fidelity. Preliminary results suggest that the origins of this behavior lie in a flexible binding pocket. A key goal of this project is to understand how this flexible active site is able to adapt to substrates of different shapes and functionalities and stereoselectively transform these substrates. More specifically, the team seeks to elucidate the mechanisms by which such biocatalysts effectively make two ‘stereochemical choices’, namely enantiodiscrimination (choosing one of two rapidly equilibrating enantiomers-sets first stereocenter) and facial discrimination (delivering a nicotinamide-derived hydride equivalent to one of two diastereofaces-sets second stereocenter).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

大多数高药用药物和农用化学品都具有非常特殊的结构，因此酶可以有效地产生这种结构。参与生产一类称为紫杉烷的精细化学物质的酶的活性，这些化合物具有抗癌特性，其中一种紫杉醇目前用于治疗转移性乳腺癌。和指导还计划与一所拥有多元化学生群体的大型公立高中的科学系合作，培养生物分子工程、化学和结构生物学领域的本科生和研究生。分子动力学模拟和 X 射线晶体学工具将指导两种有前途的烟酰胺依赖性氧化还原酶的定向进化，重点是对这些酶进行工程改造，使种族底物可以有效地去消旋化并转化为主要的一种。通过动态还原动力学解析四种可能的立体异构体的目标转化将为天然产物合成、化学生物学和药物化学提供一系列有价值的构建模块，这些构建模块可用于紫杉烷家族的化疗药物。在之前的工作中，该团队已经证明梭菌酶（CaADH）表现出显着的底物混杂性，同时表现出相当大的立体化学保真度。这种行为的起源在于灵活的结合口袋，该项目的一个关键目标是了解这种灵活的活性位点如何能够适应不同形状和功能的基材，并立体选择性地改变这些基材。阐明此类生物催化剂有效地做出两种“立体化学选择”的机制，即对映体歧视（选择两种快速平衡的对映异构体中的一种 - 设置第一个立体中心）和面部歧视（提供烟酰胺衍生的氢化物相当于两个非对映面集第二立体中心之一）。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。