文摘
In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed ofthe 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 MccB17 synthetase as fusions tomaltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminarycharacterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initialcyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase.We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as aconformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as theinitial site of substrate recognition. Heterocyclization activity was reconstituted only by combining allthree subunits, demonstrating that each protein is required for heterocycle formation. Titration assaysindicate that the subunits bind to each other with at least micromolar affinities, although McbD affordsactivity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147Amutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complexformation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibitsreconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A modelfor synthetase activity is proposed.
