Phylogeny‐guided Characterization of Bacterial Hydrazine Biosynthesis Mediated by Cupin/methionyl tRNA Synthetase‐like Enzymes

Abstract

Cupin/methionyl‐tRNA synthetase (MetRS)‐like didomain enzymes catalyze nitrogen‐nitrogen (N–N) bond formation between Nw‐hydroxylamines and amino acids to generate hydrazines, key biosynthetic intermediates of various natural products containing N–N bonds. While the combination of these two building blocks leads to the creation of diverse hydrazine products, the full extent of their structural diversity remains largely unknown. To explore this, we herein conducted phylogeny‐guided genome‐mining of related hydrazine biosynthetic pathways consisting of two enzymes: flavin‐dependent Nw‐hydroxylating monooxygenases (NMOs) that produce Nw‐hydroxylamine precursors and cupin/MetRS‐like enzymes that couple the Nw‐hydroxylamines with amino acids via N–N bonds. A phylogenetic analysis identified the largely unexplored sequence spaces of these enzyme families. The biochemical characterization of NMOs demonstrated their capabilities to produce various Nw‐hydroxylamines, including those previously not known as precursors of N–N bonds. Furthermore, the characterization of cupin/MetRS‐like enzymes identified five new hydrazine products with novel combinations of building blocks, including one containing non‐amino acid building blocks: 1,3‐diaminopropane and putrescine. This study substantially expanded the variety of N–N bond forming pathways mediated by cupin/MetRS‐like enzymes.