Project Summary/Abstract Late-stage functionalization is a powerful strategy used in nature and in synthetic chemistry to expand structural complexity and diversity. In natural product biosynthesis, this strategy is exemplified by diverse reactions catalyzed by metalloenzymes. Among them, non-heme-iron- and 2-oxoglutarate-dependent (Fe/2OG) enzymes have been shown to perform a staggering array of transformations including carbo- and hetero-cyclization, halogenation, hydroxylation, olefination, and rearrangement. The catalytic diversity makes Fe/2OG enzymes an attractive target for biocatalyst development, organic synthesis, and other applications. However, despite the prevalence of Fe/2OG enzymes in all kingdoms of life (>160,000 encoded Fe/2OG enzyme genes in genomic data), efficient and precise strategies to annotate Fe/2OG enzyme functions are still lacking. Cyclopropane and aza-cyclopropane (aziridine) are unique three-membered carbo-/hetero-cycles, and are important pharmacophores widely used in drugs. In addition, the isocyano (isonitrile) group is found in hundreds of secondary metabolites with roles in metal trafficking. As demonstrated in the preliminary results, our combined bioinformatics and retro-biosynthetic analysis has identified Fe/2OG cyclopropanases, aziridinases and isonitrilases from thousands of functionally unannotated Fe/2OG enzymes. In this project, we will continue to apply this strategy to identify enzymes with these targeted activities. In cyclopropanation and aziridination, these newly discovered Fe/2OG enzymes and the related Fe/2OG hydroxylases will be subjected to mechanistic studies using a multifaceted approach consisting of molecular probe design/synthesis, product analysis, spectroscopic (EPR and Mössbauer) characterization, transient enzyme kinetics, and protein structure prediction and computational modeling. The acquired mechanistic insights, together with bioinformatics and retro-synthetic analysis, will be used to guide Fe/2OG enzyme functional (re)assignment in genomic data, to formulate effective strategies for conversion of hydroxylases to cyclopropanases or aziridinases, and to enable alternative carbo- and hetero-cycle formations. In the isonitrilase project, we will define factors that govern substrate selectivity of these enzymes, elucidate product structures, innovate a versatile method to prepare isonitrile-containing molecules, and assess their metal-binding properties.