acrB Mutation Located at Carboxyl-terminal Region of Gyrase B Subunit Reduces DNA Binding of DNA Gyrase

Mutations that exhibit susceptibility to acriflavine have been isolated and classified as acrmutations in Escherichia coli. We cloned theacrB gene, which has been identified as a mutation of thegyrB gene, and found a double point mutation altering two consecutive amino acids (S759R/R760C) in the COO...

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Bibliographic Details
Published in:The Journal of biological chemistry 1997-05, Vol.272 (20), p.13302-13308
Main Authors: Funatsuki, Kenzo, Tanaka, Reiji, Inagaki, Shuichiro, Konno, Haruyoshi, Katoh, Kenji, Nakamura, Hakobu
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacterial Proteins - genetics
Carrier Proteins
Cloning, Molecular
DNA Gyrase
DNA Topoisomerases, Type II - genetics
DNA Topoisomerases, Type II - metabolism
DNA, Bacterial - genetics
DNA, Bacterial - metabolism
Escherichia coli
Escherichia coli - genetics
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
Membrane Proteins - genetics
Molecular Sequence Data
Multidrug Resistance-Associated Proteins
Mutation
Protein Binding
Sequence Alignment
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Summary:Mutations that exhibit susceptibility to acriflavine have been isolated and classified as acrmutations in Escherichia coli. We cloned theacrB gene, which has been identified as a mutation of thegyrB gene, and found a double point mutation altering two consecutive amino acids (S759R/R760C) in the COOH-terminal region of the gyrase B subunit. The mutant B subunit was found to associate with the A subunit to make the quaternary structure, and the reconstituted gyrase showed an 80-fold reduction of specific activity in DNA supercoiling assay; the sensitivity to acriflavine was not different in the same unit of wild-type and mutant gyrases. The mutant enzyme retained intrinsic ATPase activity, but DNA-dependent stimulation was observed infrequently. A gel shift assay showed that acriflavine inhibited the DNA binding of gyrase. The acrBmutation also reduced significantly the DNA binding of gyrase but did not change the sensitivity to acriflavine. These results revealed that the acrB mutation is related to the inhibitory mechanism of acriflavine; and the acriflavine sensitivity of the mutant, at leastin vitro, is caused mainly by reduction of the enzyme activity. Further, our findings suggest that the COOH-terminal region of the B subunit is essential for the initial binding of gyrase to the substrate DNAD87842.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.272.20.13302