Expressed protein ligation to probe regiospecificity of heterocyclization in the peptide antibiotic microcin B17

Background: The Escherichia coli peptide antibiotic microcin B17 (MccB17) contains thiazole and oxazole heterocycles derived from a distributive yet directional cyclization of cysteines and serines in the McbA precursor catalyzed by MccB17 synthetase. Whether the formation of upstream rings potentiates downstream heterocyclization has not been previously determined. Results: McbA fragments (46-61 residues) containing glycine substitutions or homocysteine at select upstream cysteine or serine sites were assembled using expressed protein ligation (EPL). Most of these substrates were only partially cyclized by MccB17 synthetase, in contrast to the efficient processing of wild-type McbA(1-61). Homocysteine was not processed to the six-membered heterocycle. Conclusions: The formation of upstream rings in McbA potentiates the cyclization of carboxy-terminal cysteines and serines, probably by selecting against unfavorable substrate conformations. EPL allows structure-function analysis including unnatural amino acid placements to probe the regiospecificity and chemoselectivity of post-translational heterocyclization during antibiotic maturation.