Genome mining for the discovery of peptide halogenases and their biochemical characterization.

While halogenation is one of the most versatile C-H functionalization strategy, regiospecific halogenation of peptides and proteins is outside the purview of traditional chemical catalysis. Enzymes that participate in the biosynthesis of ribosomally synthesized and post-translationally modified peptides and proteins can bridge this gap and offer a biocatalytic route for residue-specific incorporation of halogen handles onto amino acid side chains. Protocols described herein provide a guided approach for the discovery of peptide halogenases in the context of natural product biosynthetic gene clusters, and the preliminary reconstitution of their activity using a bacterial heterologous host. Also described are mass spectrometry-based analytical procedures and data analysis workflows that allow for deconvolution of halide specificity and preliminary insights into peptidic natural product biosynthetic schemes. As the available genomic data expands at a rapid rate, the methodology described here will enable the discovery and characterization of new halogenases that can be valuable partners in chemoenzymatic diversification of peptides and proteins.