The DNA cleavage reaction of DNA gyrase. Comparison of stable ternary complexes formed with enoxacin and CcdB protein.
The potent synthetic fluoroquinolones and the natural CcdB protein encoded by the F plasmid both inhibit bacterial growth by attacking DNA gyrase and by stimulating enzyme-induced breaks in bacterial DNA. The cleavage mechanisms of these structurally diverse compounds were analyzed by purifying and characterizing stable ternary complexes of enoxacin and CcdB protein with gyrase bound to a strong gyrase binding site from bacteriophage Mu. Three differences between enoxacin- and CcdB-derived complexes were discovered. 1) Enoxacin binds to the DNA active site and alters the breakage/reunion activity of the enzyme. CcdB binds gyrase-DNA complexes but does not influence enzymatic activity directly. 2) Complexes that produce DNA cleavage with enoxacin are reversible, whereas similar complexes made with CcdB protein are not. 3) Enoxacin stimulates cleavage of both relaxed and supercoiled forms of DNA in the absence of ATP, whereas CcdB induces cleavage only after many cycles of ATP-dependent breakage and reunion. These differences in mechanisms can be explained by a model in which enoxacin induces formation of a novel "cleavable" complex, whereas CcdB protein traps a very rare "cleaved" conformation of the enzyme.
Scheirer
K E
KE
Department of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, Alabama 35294, USA.
Higgins
N P
NP
D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D010583:Bacteriophage mu / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D003603:Cytotoxins / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D027081:DNA Gyrase; D017931:DNA Primers; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification / Q000378:metabolism*; D015365:Enoxacin / Q000378:metabolism*; D046911:Macromolecular Substances; D008961:Models, Structural; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D003603:Cytotoxins; D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D015365:Enoxacin; D004247:DNA; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II