Cofactor Requirements and Reconstitution Of Microcin B17 Synthetase:  A Multienzyme Complex that Catalyzes the Formation of Oxazoles and Thiazoles in the Antibiotic Microcin B17

Abstract

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 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 McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/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.

Identification of a Streptolysin S-Associated Gene Cluster and Its Role in the Pathogenesis ofStreptococcus iniaeDisease

“…McbC is involved in the posttranslational modification of McbA, to produce the mature bacteriocin microcin B17, and uses flavin mononucleotide (FMN) as a cofactor (36). A conserved domain in the SagB ORF of S. iniae (positions 125 to 298) was identified by using the conserved domain database (RPS-BLAST) in GenBank.…”

“…Furthermore, the FMN-binding regions of T. thermophilus NO (24) are potentially conserved in other flavoproteins (47) as well as SagB. The amino acid sequences of the SagB products also share weak homology with McbC of E. coli (20 (36). The bacteriocin epidermin, produced by Staphylococcus epidermidis, also requires the enzymatic action of a flavoprotein, EpiD, for posttranslational modification (28).…”

Patellamide A and C biosynthesis by a microcin-like pathway inProchloron didemni, the cyanobacterial symbiont ofLissoclinum patella

“…Alternatively, the ATP-binding region could have an as-yet-unknown function. For example, it is known that the microcin B17 heterocyclization complex includes an ATP-requiring enzyme, McbD, which is of unknown function (46). PatD1 does not show significant sequence homology to McbD, but it is often the case in microcin machinery that distantly related peptides serve similar functions.…”

Structure of Microcin B-Like Compounds Produced by Pseudomonas syringae and Species Specificity of Their Antibacterial Action

“…coli cells producing microcin B contain plasmids carrying a 7-gene mcb operon. The mcbA gene encodes a precursor peptide; the mcbBCD genes encode subunits of the microcin B synthase, an enzyme responsible for the synthesis of oxazole and thiazole heterocycles (8,9); the mcbEF genes encode an export pump; and mcbG encodes a pentapeptide repeat protein that binds to a microcin B target, the DNA gyrase, rendering it resistant to microcin B inhibition (10). The microcin B precursor, the 69-amino-acidlong McbA, contains an N-terminal leader (amino acids 1 to 26) that is recognized by the McbBCD complex.…”

“…The microcin B precursor, the 69-amino-acidlong McbA, contains an N-terminal leader (amino acids 1 to 26) that is recognized by the McbBCD complex. After binding to the leader, the McbBCD synthase moves in the C-terminal direction, converting the GlySer and GlyCys McbA dipeptides into oxazole and thiazole heterocycles, respectively (9). Two McbA tripeptides, GlySerCys and GlyCysSer, are converted into oxazole-thiazole and thiazole-oxazole bis-heterocycles (site A and site B), thought to be important for microcin B activity (11).…”