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

Background: The Escherichia coli peptide antibiotic microcin B17 (MccB17) c ontains thiazole and oxazole heterocycles derived from a distributive yet d irectional cyclization of cysteines and serines in the McbA precursor catal yzed by MccB17 synthetase. Whether the formation of upstream rings potentia tes downstream heterocyclization has not been previously determined. Results: McbA fragments (46-61 residues) containing glycine substitutions o r homocysteine at select upstream cysteine or serine sites were assembled u sing expressed protein ligation (EPL). Most of these substrates were only p artially cyclized by MccB17 synthetase, in contrast to the efficient proces sing of wild-type McbA(1-61). Homocysteine was not processed to the six-mem bered heterocycle. Conclusions: The formation of upstream rings in McbA potentiates the cycliz ation of carboxy-terminal cysteines and serines, probably by selecting agai nst unfavorable substrate conformations. EPL allows structure-function anal ysis including unnatural amino acid placements to probe the regiospecificit y and chemoselectivity of post-translational heterocyclization during antib iotic maturation.