Mechanisms of Enzymatic Vinyl Isonitrile Formation and Reprogramming of Isonitrilases and Iron/2-Oxoglutarate Desaturases

项目摘要/摘要 异腈酶参与从伯胺和碳水化合物的羰基部分构建三键。 磷酸盐。非血红素铁和依赖2-(氧)戊二酸(Fe/2OG)的酶催化了一系列令人眼花缭乱的 转化，包括卤化、环化、脱氢、内过氧化和 脂肪族碳中心的立体反转。在40多种含有天然产物的分离的乙烯基异腈中， 一种通用的异腈酶两步酶转化通用方法 建议使用Fe/2OG依赖的烯烃形成酶来安装这些官能团。然而， 对这些转变的机械性轻描淡写仍有待阐明。一种化学和生物合成材料 了解这些反应机理可以使异腈酶和Fe/2OG酶重新编程。 作为生物催化剂，用于生产具有更高生物活性的新化合物。最近取得了进展， Fe/2OG酶在体外催化环氧化和去饱和化及异腈酶活力重建 建议研究异腈和脱羧基辅助的烯烃生成反应。 大黄素和白藜芦素的生物合成。我们将使用一种综合的方法来提供分子 了解酶的作用机制，并在某些情况下评估底物的灵活性和重新编程 相关酶的定向进化。

Project Summary/Abstract Isonitrilase is involved in constructing a triple bond from a primary amine and a carbonyl moiety of carbohydrate phosphate. Non-heme iron and 2-(oxo)glutarate-dependent (Fe/2OG) enzymes catalyze a bewildering array of transformations, which include halogenations, cyclizations, dehydrogenations, endoperoxidation and stereoinversion of aliphatic carbon centers. In more than 40 isolated vinyl isonitrile containing natural products, a universal approach involving a consecutive two-step enzymatic transformation by an isonitrilase and an Fe/2OG dependent alkene forming enzyme is proposed to install these functional groups. However, the mechanistic understating of these transformations remains to be elucidated. A chemical and biosynthetic understanding of these reaction mechanisms could enable reprograming of isonitrilase and Fe/2OG enzymes as biocatalysts for producing new compounds with improved bioactivity. Having recently made progress toward Fe/2OG enzyme catalyzed epoxidation and desaturation, and isonitrilase activity reconstitution in vitro, we propose to study the isonitrile and decarboxylation-assisted alkene formation reactions on the pathway to rhabduscin and paerucumarin biosyntheses. We will use an integrated approach to provide molecular understanding of the enzyme mechanisms and, in select cases, evaluate the substrate flexibility and reprogram the related enzymes by directed evolution.