Bacterial Biosynthesis of Nitrile-Containing Natural Products: Basis for Recognition of Diversified Substrates

Abstract

Nitrile-containing natural products, despite being a limited group of secondary metabolites, display remarkable structural and functional diversities. Aldoxime formation represents a crucial step in nitrile installation via the aldoxime-nitrile pathway although structural information regarding aldoxime formation is extremely limited. Here, we report the isolation of a nitrile compound 6-dimethylallylindole-3-acetonitrile (6-DMAIAN) and identify the aldoxime-forming enzyme gene diatB as a robust reporter for bacterial nitrile biosynthesis. We characterize the flavin-dependent monooxygenase DiatB and provide structural and mechanistic insights into the structural parameters dictating its substrate compatibilites. This enzyme initiates a nucleophilic attack on the amino group of the substrate 6-dimethylallyl-l-tryptophan (6-DMAT), resulting in formation of a transient aldoxime that precedes nitrile installation. Moreover, the DiatB recognition motif is elucidated shedding light on its substrate flexibility. We also apply bioinformatics analysis to examine the distribution and diversity of functional DiatB homologues across an array of potential nitrile-forming organisms. Given the activity of DiatB and its prevalence in secondary metabolite biosynthesis, our results provide important insight into what is, arguably, the most crucial and pivotal step in nitrile biosynthesis; these findings also suggest a promising enzymatic tool for nitrile drug design.