TY - JOUR
T1 - Cofactor requirements and reconstitution of Microcin B17 synthetase
T2 - A multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic Microcin B17
AU - Milne, Jill C.
AU - Sinha Roy, Ranabir
AU - Eliot, Andrew C.
AU - Kelleher, Neil L.
AU - Wokhlu, Anita
AU - Nickels, Bryce
AU - Walsh, Christopher T.
PY - 1999/4/13
Y1 - 1999/4/13
N2 - In the maturation of the Escherichia coli antibiotic Microcin B 17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric 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 MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration 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 a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbD(D147A) mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbB(C181A/C184A) double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.
AB - In the maturation of the Escherichia coli antibiotic Microcin B 17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric 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 MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration 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 a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbD(D147A) mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbB(C181A/C184A) double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.
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U2 - 10.1021/bi982975q
DO - 10.1021/bi982975q
M3 - Article
C2 - 10200165
AN - SCOPUS:0039130745
SN - 0006-2960
VL - 38
SP - 4768
EP - 4781
JO - Biochemistry
JF - Biochemistry
IS - 15
ER -