Rs. Roy et al., Expressed protein ligation to probe regiospecificity of heterocyclization in the peptide antibiotic microcin B17, CHEM BIOL, 6(11), 1999, pp. 789-799
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.