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Mutation of the N‐terminal proline 9 of BLMA from Streptomyces verticillus abolishes the binding affinity for bleomycin
Author(s) -
Kumagai Takanori,
Hibino Ryu,
Kawano Yoshiaki,
Sugiyama Masanori
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)00478-0
Subject(s) - proline , bleomycin , mutation , chemistry , terminal (telecommunication) , streptomyces , stereochemistry , microbiology and biotechnology , genetics , biology , biochemistry , amino acid , gene , bacteria , telecommunications , chemotherapy , computer science
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, 176–184], 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.