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

大多数药品和农用化学品都被称为精细化学品。这些分子具有非常特殊的结构，这赋予了它们有益的特性。酶在产生这种结构方面非常有效。该项目的目标是开发一种策略来设计和调整酶的活性，这些酶参与生产一类被称为紫杉烷的精细化学品。这些化合物具有抗癌特性。紫杉醇是紫杉烷中的一种，目前被用于治疗转移性乳腺癌。该项目将包括在生物分子工程、化学和结构生物学方面对本科生和研究生进行教育和指导。还计划与一所拥有不同学生群体的大型公立高中的科学系合作。这些努力将培养一支劳动力队伍，以支持充满活力的生物经济。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的氧化还原酶的定向进化。重点是工程这些酶的反应，其中外消旋底物可以有效地去消旋，并通过动态还原动力学分辨率转化为四种可能的立体异构体之一。有针对性的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。几个构建块是为化疗药物的紫杉烷家族，有用的抗癌药物。在之前的工作中，该团队已经证明梭状菌酶（CaADH）表现出显著的底物混杂性，同时表现出相当大的立体化学保真度。初步结果表明，这种行为的起源在于一个灵活的装订袋。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底物，并对这些底物进行立体选择性转化。更具体地说，该团队试图阐明这种生物催化剂有效地进行两种“立体化学选择”的机制，即对映体辨别（从两个快速平衡的对映体中选择一个-设置第一个立体中心）和面辨别（提供相当于两个非对映体之一的烟酰胺衍生氢化物-设置第二个立体中心）。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.