From peptide precursors to oxazole and thiazole-containing peptide antibiotics: Microcin B17 synthase

Science

Volumn 274, Issue 5290, 1996, Pages 1188-1193

  Li, Yue Ming ^a   Milne, Jill C ^a   Madison, Lara L ^a   Kolter, Roberto ^a   Walsh, Christopher T ^a  

^a HARVARD MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL DNA; BLEOMYCIN A2; GYRASE INHIBITOR; MICROCIN B17; OXAZOLE; POLYPEPTIDE ANTIBIOTIC AGENT; PROTEIN PRECURSOR; SYNTHETASE; THIAZOLE; UNCLASSIFIED DRUG;

ARTICLE; ENZYME PURIFICATION; ESCHERICHIA COLI; OPERON; PRIORITY JOURNAL; PROTEIN BINDING;

ADENOSINE TRIPHOSPHATE; ANTI-BACTERIAL AGENTS; BACTERIAL PROTEINS; BACTERIOCINS; DNA TOPOISOMERASES, TYPE II; ENZYME INHIBITORS; ESCHERICHIA COLI; MOLECULAR WEIGHT; MULTIENZYME COMPLEXES; OPERON; OXAZOLES; OXIDATION-REDUCTION; OXYGEN; PROTEIN PRECURSORS; PROTEIN PROCESSING, POST-TRANSLATIONAL; SUBSTRATE SPECIFICITY; THIAZOLES;

BACTERIA (MICROORGANISMS); ESCHERICHIA COLI;

EID: 0029923954     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5290.1188     Document Type: Article

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31. 1 transduction of zba-315::kan, ΔhtpG::lacZ from JCB42 [J. C. A. Bardwell and E. A. Craig, J. Bacterial. 170, 2977 (1988)]. The htpG knockout strain MC4100 ΔhtpG and control strain MC4100 were transformed with plasmid pPY113. These strains were assayed as described in Fig. 3B except that the McbA-β-Gal concentration was 30 μM.
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56. All four peptides were purified by HPLC [Bio-Rad Preparative C18 column, with solvent A: 0.1% trifluoroacetic acid (TFA) in water, and solvent B: 0.1% TFA in acetonitrile] and the structure confirmed by mass spectrometry. The concentration of the peptides was determined by amino acid analysis.
57. The ZK4(pPY113) strain was used for purification of MccB17 synthase. An overnight culture grown in LB media containing ampicillin (Amp) was diluted 500-fold in M63 medium containing Amp and grown at 37°C for 20 to 24 hours. The cells were pelleted by centrifugation (5000g) and lysed with a French pressure cell. The supernatant after 105,000g centrifugation was used for purification by phenyl-Sepharose, DEAE-Sepharose, Sephacryl S-200, and MonoQ (Pharmacia). The activity was monitored by protein immunoblot analysis as described in Fig. 3. with McbA-β-Gal as a substrate.
58. 2] four times and collected by centrifugation. SDS sample buffer was added to the complex, and the supernatants were subjected to protein immunoblot analysis.
59. 2, 1 mM ATP, 1 mM dithiothreitol, and purified MccB17 synthase (0.24 mg/ml). The reactions were carried out at 37°C and stopped by injection onto HPLC (Vydac C18 column, 300 Å, solvent A: 0.1% TFA in water, and solvent B: 0.09% TFA in acetonitrile). McbA(1-46) was eluted around 33% solvent B. The 0- and 45-min reaction mixtures showed the same elution patterns, a peak at 33% solvent B and other protein peaks after >45% solvent B. HPLC fractions were analyzed by protein immunoblot with anti-MccB17 and only the peak (33% solvent B) for the 45-min reaction was antibody-positive (18).
60. 2-terminal sequencing, amino acid analysis, and MS analysis; S. Hecht (University of Virginia) for aminoethyl bithiazole carboxylic acid; M. Bollinger (Pennsylvania State University) and S Fisher and D. McCafferty of this laboratory for their help and suggestions regarding the anaerobic reactions. We also thank other members of the C.T.W. laboratory for suggestions concerning this manuscript. Supported by NIH grant GM20011 (C.T.W.) and NSF grant MCB9207323 (R.K.). J.C.M. is supported by a postdoctoral fellowship (grant PF4332) from the American Cancer Society, and L.L.M. is supported by NIH training grants 5T32GM07306-19 and 5T32Al07410-03.