Posttranslational heterocyclization of cysteine and serine residues in theantibiotic Microcin B17: Distributivity and directionality

To produce the antibiotic Microcin B17, four Cys and four Ser residues are converted into four thiazoles and four oxazoles by the three subunit Microc in B17 synthetase. High-resolution mass spectrometry (MS) was used to monit or the kinetics of posttranslational heterocyclic ring formation (-20 Da pe r ring);and demonstrated the accumulation of all intermediates, from one to seven rings, indicating distributive processing. All of the intermediates could be converted by the enzyme to the eight ring product. Enzymatic chemo selectivity (Cys vs Ser cyclization rates) was assessed using iodoacetamido -salicylate to alkylate unreacted cysteines (+193 Da) in the 8 kDa biosynth etic intermediates; three of the first four rings formed were thiazoles, an d by the five ring stage, all four of the cysteines had been heterocyclized while three of the original four serines remained uncyclized. Finally, tan dem MS using a 9.4 T Fourier transform instrument with electrospray ionizat ion was used to elaborate the major processing pathway: the first two rings formed are at the most amino proximal sites (Cys(41) then Ser(40)) followe d by the remaining three cysteines at positions 48, 51, and 55. The cycliza tion of serines at positions 56, 62, and 65 then follows, with Ser(62) and Ser(65) the last to heterocyclize and the first of these at a slower rate. Thus, despite free dissociation of intermediates after each of seven ring-f orming catalytic cycles, there is an overall directionality of ring formati on from N-terminal to C-terminal sites. This remarkable regioselectivity is determined more by the substrate than the enzyme, due to a combination of (1) initial high-affinity binding of the posttranslational catalyst to the N-terminal propeptide of substrate 88mer, and (2) a chemoselectivity for th iazole over oxazole formation. This mechanism is consistent with antibiotic biosynthesis in vivo, yielding microcin with six, seven, and eight rings, all with bioactivity.