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

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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.