Nucleic Acids Research, 2002, Vol. 30, No. 10 2144-2153
© 2002 Oxford University Press
Functional characterisation of mycobacterial DNA gyrase: an efficient decatenase
1Department of Microbiology and Cell Biology, 2Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560 012, India and 3Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 054, India
A rapid single step immunoaffinity purification procedure is described for Mycobacterium smegmatis DNA gyrase. The mycobacterial enzyme is a 340 kDa heterotetrameric protein comprising two subunits each of GyrA and GyrB, exhibiting subtle differences and similarities to the well-characterised Escherichia coli gyrase. In contrast to E.coli gyrase, the M.smegmatis enzyme exhibits strong decatenase activity at physiological Mg2+ concentrations. Further, the enzymes exhibited marked differences in ATPase activity, DNA binding characteristics and susceptibility to fluoroquinolones. The holoenzyme showed very low intrinsic ATPase activity and was stimulated 20-fold in the presence of DNA. The DNA-stimulated ATPase kinetics revealed apparent K0.5 and kcat of 0.68 mM and 0.39 s–1, respectively. The dissociation constant for DNA was found to be 9.2 nM, which is 20 times weaker than that of E.coli DNA gyrase. The differences between the enzymes were further substantiated as they exhibited varied sensitivity to moxifloxacin and ciprofloxacin. In spite of these differences, mycobacterial DNA gyrase is a functionally and mechanistically conserved enzyme and the variations in activity seem to reflect functional optimisation for its physiological role during mycobacterial genome replication.
* To whom correspondence should be addressed at: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India. Tel: +91 80 360 0668; Fax: 91 80 360 2697; Email: vraj{at}mcbl.iisc.ernet.in The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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