Mutational analysis of posttranslational heterocycle biosynthesis in the gyrase inhibitor microcin B17: distance dependence from propeptide and tolerance for substitution in a GSCG cyclizable sequence.
Microcin B17 (MccB17) is a peptidyl antibiotic that is secreted in stationary phase by several strains of Escherichia coli. The antibiotic efficacy of this polypeptide depends on the posttranslational modification of eight cysteine and serine residues to thiazoles and oxazoles, respectively, within the 69 aa McbA structural gene product. Mono- and bisheterocycle formation is mediated by MccB17 synthetase, an enzyme complex composed of three proteins: McbB, -C, and -D. After substrate processing, an N-terminal 26 aa propeptide sequence is cleaved to afford the mature antibiotic. A method for the overexpression and rapid purification of microcin synthetase has been developed using a calmodulin-binding peptide tag. The determinants of substrate recognition and synthetase-mediated heterocycle formation were investigated by a systematic evaluation of 15 McbA1-46 analogues representing minimal substrates containing the first bisheterocyclization site (Gly39-Ser40-Cys41-Gly42) and variants thereof. Each substrate analogue was overexpressed and affinity-purified as fusions to maltose-binding protein, incubated with purified synthetase, and evaluated for processing by Western blots, UV spectroscopy, and mass spectrometry. Insights gained into the process of enzymatic heterocycle formation from cysteine and serine residues are discussed, including the distance dependence of the first cyclized residue from the propeptide and the local sequence context at the cyclizable sites. A model for McbA substrate recognition and processing by MccB17 synthetase is proposed.
Sinha Roy
R
R
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Belshaw
P J
PJ
Walsh
C T
CT
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins; D001430:Bacteriocins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D059005:Topoisomerase II Inhibitors
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D001430:Bacteriocins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009097:Multienzyme Complexes; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D059005:Topoisomerase II Inhibitors; C461599:maltose transport system, E coli; C102057:microcin B17 synthase; C020411:microcin; C011080:polyglycine