Production of bleomycin N-acetyltransferase in Escherichia coli and Streptomyces verticillus.
Bleomycin-producing Streptomyces verticillus ATCC 15003 has two bleomycin resistance genes, designated blmA and blmB. Bleomycin N-acetyltransferase, encoded by blmB, was overproduced in Escherichia coli as a protein fused to the maltose-binding protein. The protein (fBAT), purified to homogeneity after digestion of the fusion product with blood coagulation factor Xa protease, had an additional 6 N-terminal amino acid residues, but retained its bleomycin-acetylating activity, as did the entire fusion protein. The K(m) and Vmax values of purified fBAT for the substrate bleomycin were 13.0 microM and 3.4 nmol [corrected] min-1 ml-1, respectively. The optimal pH for the acetylating activity was 6.0 in 10 mM phosphate buffer. The molecular mass and pI value of fBAT were estimated by polyacrylamide gel electrophoresis to be about 34500 and 6.13, respectively. An anti-fBAT monoclonal antibody was generated and used to show that bleomycin N-acetyltransferase is expressed simultaneously with bleomycin production in S. verticillus.
Matsuo
H
H
Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, Japan.
Mochizuki
H
H
Davies
J
J
Sugiyama
M
M
D000123:Acetyltransferases / Q000096:biosynthesis*; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001761:Bleomycin / Q000378:metabolism*; D004352:Drug Resistance, Microbial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000276:immunology
D000900:Anti-Bacterial Agents; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D001761:Bleomycin; C087954:bleomycin N-acetyltransferase; D000123:Acetyltransferases