Abstract
The mechanism of inhibition of DNA gyrase by cyclothialidine, a novel gyrase inhibitor isolated from Streptomyces filipinensis NR0484, has been studied further by using [14C]benzoylcyclothialidine and a reconstituted Escherichia coli gyrase system consisting of the A subunit, the B subunit and relaxed ColE1 DNA. The mechanism of inhibition was also studied with the 43-kDa N-terminal fragment of the B subunit. The [14C]benzoylcyclothialidine could bind to the B subunit alone but not to the A subunit nor to the plasmid DNA alone. Furthermore, the compound also bound to the 43-kDa N-terminal fragment of the B subunit. Scatchard analysis of [14C]benzoylcyclothialidine binding to DNA gyrase showed that the binding affinity of the compound increased, depending on the assembly of the gyrase (A2B2). DNA complex. This suggests that the binding site of cyclothialidine on the B subunit or its vicinity causes a conformational change during the assembly of the gyrase.DNA complex (increase in affinity: B-->A2B2-->A2B2.DNA). Furthermore, displacement curves of [14C]benzoylcyclothialidine binding by nonlabeled cyclothialidine, ATP analogues, and coumarin antibiotics indicated that cyclothialidine, coumarins, and ATP share a common (or overlapping) site of action on the B subunit of DNA gyrase; however, the microenvironment of the binding sites may differ.
Highlights
From the Nippon Roche Research Center, Kamakura 247, Japan and §F
This suggests that the binding site of cyclothialidine on the B subunit or its vicinity causes a conformational change during the assembly of the gyrase·DNA complex
We showed that cyclothialidine binds to the B subunit and inhibits the ATPase activity of DNA gyrase [23]
Summary
Materials-Cyclothialidine was purified from a culture broth of S. filipinensis NR0484 having a >98% purity detected by HPLC analysis [21]. Purification ofthe 43-kDa N-terminal Fragment ofthe DNA Gyrase B Subunit-The 43-kDa protein was purified from E. coli cells containing plasmid pAJl essentially as described by Jackson et al [27]. Fractions containing the protein fragment were identified by SDS-polyacrylamide gel electrophoresis, pooled, and dialyzed against 50 mM Tris-HCI (pH 8.0), 100 mM KCI, 5 mM dithiothreitol, 1 mM EDTA, 10% (w/v) glycerol, frozen in liquid nitrogen, and stored at -80 DC [28]. Reactions (400 ul) were carried out similar to that of the supercoiling assay but without the addition of ATP; the reaction mixture contained an appropriate amount of DNA gyrase and radioactive ligand in standard buffer (50 mM Tris-HCI (pH 8.0), 20 mM KCI, 10 mM MgC12 , 1 mM EDTA, and 1 mM dithiothreitol).
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