Investigation on the Biosynthesis and Structural Diversity of Biarylitides Uncovers Novel Biosynthetic Paradigm

Abstract

Ribosomally synthesised and post-translationally modified peptides (RiPPs) constitute a major group of natural products. Biarylitides, a novel class of RiPPs first isolated from the bacterial genus <em>Planomonospora</em>, are biaryl-linked tripeptides synthesised by only two genes: <em>bytA</em>, which encodes a minimal five-amino acid precursor with a conserved MxYxH motif, and <em>bytO</em>, which encodes a cytochrome P450 monooxygenase (P450) that forms a C-C biaryl crosslink between the tyrosine and histidine aromatic side chains within the peptide. This thesis presents several advances in the understanding of biarylitide biosynthesis and diversity. A detailed characterisation of the BytO homologue P450_Blt from <em>Micromonospora</em> sp. MW-13 unveiled a high degree of substrate flexibility, as well as selective formation of C-N crosslinks, even when tryptophan replaces the canonical histidine in the peptide substrate. Crystal structure analysis of P450<em>Blt</em> in complex with its substrate MRYLH identified key residues controlling substrate coordination and reaction specificity. The central discovery of this work revealed that rufomycin biosynthesis contains an unprecedented integration of ribosomal and non-ribosomal peptide pathways, thereby solving the mystery of nitrotyrosine formation. In this pathway, a biarylitide-like precursor peptide MRYLH undergoes selective tyrosine nitration catalysed by the BytO homologue RufO. Following proteolytic cleavage of the nitrated peptide, the released nitrotyrosine is incorporated by a non-ribosomal peptide synthetase to yield the potent antibiotic rufomycins. This neofunctionalised biarylitide machinery established a new paradigm for producing non-proteinogenic amino acids through RiPP pathways using peptides as templates. To explore the broader diversity of biarylitide systems in nature, machine learning-based genome mining was utilised to identify 124 new gene clusters and expanded the known class to 277 members, including two clusters in the human oral microbiome-associated <em>Rothia</em> species. Heterologous expression facilitated the characterisation of four novel biarylitides, including a putative hydroxylated YVH tripeptide. A novel BytO homologue subgroup was biochemically validated to catalyse crosslinking of atypical peptide motifs (MRYWY). Furthermore, nuclear magnetic resonance analysis of <em>in vitro</em> enzymatic products verified C-N crosslink formation in MKYWH peptides, enabling prediction of the final structure of this widespread bacterial natural product. Collectively, these findings have illuminated the remarkable diversity and evolutionary plasticity of biarylitide biosynthetic systems, creating new opportunities for natural product discovery and engineered peptide modifications.