Iron and 2‐Oxoglutarate‐Dependent Isonitrile‐Forming Enzymes

Abstract

Abstract The isonitrile moiety, composed of an NC connection, is a commonly used functional group in chemical synthesis due to its highly reactive nature toward nucleophile, electrophile, and radical. Isonitrile‐containing natural products have shown diverse biological properties including virulence, antimicrobial activities, and metal acquisition. Despite its abundance in natural products, how isonitrile is introduced remains understudied. Recently, N ‐glycinyl moiety has been identified as a biosynthon for isonitrile wherein several nonheme iron and 2‐oxoglutarate‐dependent (Fe/2OG) enzymes have been identified to enable this reaction. Distinct from canonical oxygenation or halogenation catalyzed by Fe/2OG enzymes, installation of the isonitrile group from a glycinyl moiety is a four‐electron oxidation of the substrate and involves decarboxylation‐assisted desaturation, thus representing an uncommon reaction type. To elucidate plausible reaction mechanism, several Fe/2OG‐dependent enzymes involved in isonitrile‐containing peptides and polyketides were characterized. Combining mechanistic probes design, protein X‐ray crystallography, biochemical and biophysical characterizations, and mutagenesis, a plausible reaction pathway was established. Formation of isonitrile involves two sequential reactions. Following a CH bond cleavage triggered by an Fe(IV)‐oxo, a carboxyaldimine species is produced in the first reaction. In the second reaction, decarboxylation‐assisted desaturation of the carboxyaldimine intermediate affords the NC moiety.