Functional characterisation of mycobacterial DNA gyrase: an efficient decatenase.
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.
Manjunatha
U H
UH
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
Dalal
M
M
Chatterji
M
M
Radha
D R
DR
Visweswariah
S S
SS
Nagaraja
V
V
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D027081:DNA Gyrase / Q000302:isolation & purification* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D027081:DNA Gyrase