Cofactor requirements and reconstitution of Microcin B17 synthetase: A multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic Microcin B17

In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB 17) , the McbA preproantibiotic is modified post-translationally by the multime ric microcin synthetase complex (composed of the McbB, -C, and -D proteins) , which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB 17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characteri zation of each subunit reveals McbB to be a zinc-containing protein that ma y catalyze the initial cyclodehydration step, and McbC to contain flavin, c onsistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide n ow identify McbD as the initial site of substrate recognition. Heterocycliz ation activity was reconstituted only by combining all three subunits, demo nstrating that each protein is required for heterocycle formation. Titratio n assays indicate that the subunits bind to each other with at least microm olar affinities, although McbD affords activity only after the MBP tag is p roteolytically removed. Subunit competition assays with an McbD(D147A) muta nt, which yields a catalytically deficient synthetase in vivo, show it to b e defective in complex formation, whereas the McbB(C181A/C184A) double muta nt, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with Mc cB17 synthetase), does not stimulate synthetase reconstitution or heterocyc lization activity in vitro. A model for synthetase activity is proposed.