Mutation of the N-terminal proline 9 of BLMA from Streptomyces verticillus abolishes the binding affinity for bleomycin.
A gene, blmA, from bleomycin (Bm)-producing Streptomyces verticillus, encodes a Bm-binding protein, designated BLMA. The expression of BLMA conferred resistance to Bm in the Escherichia coli host, whereas a mutant protein, designated Pro-9/Leu, with the N-terminal proline 9 residue in BLMA replaced by leucine, did not. We created a fusion protein between the maltose-binding protein (MBP) and a mutant protein Pro-9/Leu/Leu with Met-94 in Pro-9/Leu replaced by leucine. Pro-9/Leu/Leu from the fusion protein, obtained by digestion with CNBr digestion, did not inhibit DNA-cleaving and antibacterial activities of Bm. Native-polyacrylamide gel electrophoresis (PAGE) and gel filtration column chromatographic analysis showed that the molecular size of Pro-9/Leu/Leu is roughly half of that of BLMA, suggesting that the mutant protein cannot form dimeric structure. Furthermore, Far-UV circular dichroism (CD) spectrum of Pro-9/Leu/Leu was quite different from that of BLMA and similar to the spectra obtained from unordered proteins [Venyaminov, S.Y. and Vassilenko, K.S. (1994) Anal. Biochem. 222, 176184], suggesting that the secondary structure of Pro-9/Leu/Leu is disrupted. These results indicate that the mutation abolishes not only dimer formation but also the secondary structure of BLMA, which results in the loss of its function as a Bm-resistance determinant.
Kumagai
T
T
Institute of Pharmaceutical Sciences, Faculty of Medicine, Hiroshima University, Japan.
Hibino
R
R
Kawano
Y
Y
Sugiyama
M
M
D000123:Acetyltransferases; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001761:Bleomycin / Q000378:metabolism*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D001761:Bleomycin; D011392:Proline; C087954:bleomycin N-acetyltransferase; D000123:Acetyltransferases; D007930:Leucine