Mechanistic Investigation of Copper-Dependent Peptide Cyclases for Macrocycle Engineering

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PROJECT SUMMARY Macrocyclic peptides are effective scaffolds for antibiotic drug discovery as they can combine the oral bioavailability and cell membrane permeability of small molecule drugs with metabolic stability and target specificity of biologics. The 14-membered bicyclic darobactin is a peptide antibiotic lead structure against Gram- negative multi-drug resistant bacteria. Darobactin is defined by two side-chain-to-side-chain-macrocyclic bonds, cyclized by a radical S-adenosylmethionine (SAM) iron-sulfur cluster enzyme. Due to synthetic challenges towards darobactin macrocyclic complexity and the anaerobic nature of its radical SAM cyclase, a biocatalytic alternative is needed to produce and diversify 14-membered bicyclic peptides in an aerobic environment. BURP domain proteins have recently been characterized from plant genomes as copper-dependent autocatalytic peptide cyclases, which catalyze the formation of darobactin-type macrocycles under aerobic conditions. BURP domain proteins constitute precursor peptides of plant ribosomally-encoded and post-translationally modified peptides (RiPPs). BURP domain precursor peptides include core peptide motifs and a C-terminal BURP domain, which catalyzes the cyclization of amino acid side chains in the core peptide in a copper-dependent reaction. BURP domain-derived peptides have diverse macrocycles: mono- and bicyclic scaffolds, 14- to 21- membered rings, and C-O, C-N- and C-C-macrocyclic bonds. Despite the chemical diversity of their cyclopeptide products, the structure and mechanism of BURP domain cyclases are completely unknown. Based on preliminary work, I hypothesize that BURP domain cyclases use a redox active copper cofactor, a radical-based mechanism, and require dioxygen for catalysis. Electron paramagnetic resonance will identify the presence of radical species and Cu(I) in BURP domain catalysis, and anaerobic reconstitution of recombinant BURP domain cyclases followed by bottom-up proteomic analysis will characterize dioxygen as a cofactor. In this proposal, the protein structures of two representative BURP domains will be determined in Specific Aim 1. Type I BURP domain cyclases encode a single core peptide within the BURP domain, represented by the bicyclase from peanut, AhyBURP. Type II BURP domain cyclases have a repetitive N-terminal core peptide domain attached to the BURP domain, and will be investigated from African clubmoss, the peptide bicyclase SkrBURP. Specific Aim 2 uses AhyBURP and SkrBURP to elucidate the catalytic mechanism of BURP domains. I also predict that BURP domain cyclases can be engineered to yield tailored macrocycles. Specific Aim 3 is to generate mimics of the antibiotic darobactin by rational design of SkrBURP, and testing the efficacy of these darobactin mimics against drug-resistant pathogenic bacteria. The proposed research of BURP domain cyclase engineering represents the possibility to generate new macrocyclic peptide libraries to address antimicrobial resistance.