Characterization of quercetin binding site on DNA gyrase

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Abstract

Gyrases are DNA topology modifying enzymes present only in prokaryotes which makes them an attractive target for antibacterial drugs. Quercetin, one of the most abundant natural flavonoids, inhibits supercoiling activity of bacterial gyrase and induces DNA cleavage. It has been generally assumed that the mechanism of flavonoid inhibition is based on interaction with DNA. We show that quercetin binds to the 24 kDa fragment of gyrase B of Escherichia coli with a KD value of 15 μM and inhibits ATPase activity of gyrase B. Its binding site overlaps with ATP binding pocket and could be competitively replaced by either ATP or novobiocin. The structural model of quercetin–gyrase complex was prepared, based on the close similarity with ATP and quercetin binding sites of the src family tyrosine kinase Hck. We propose that quercetin inhibits gyrases through two different mechanisms based either on interaction with DNA or with ATP binding site of gyrase.

Section snippets

Materials and methods

Preparation and isolation of recombinant gyrase A, B, and 24 kDa fragment of gyrase B. Coding region for the 24 kDa fragment of gyrase B was amplified by PCR from the isolated bacterial DNA. Sequences of primers for gyrA, gyrB, and 24 kDa N-terminal fragment of gyrB were: ATCGCATATGTCGAATTCTTATGACTC and ATCGGGATCCTAGCCTTCATAGTGGAAGTGGT. PCR product was cleaved with NcoI and BamHI and subcloned into the pET3a expression vector (Novagen). Production of recombinant proteins was carried out in

Inhibition of gyrase by quercetin

DNA supercoiling and relaxation experiments by gyrase were performed as a function of quercetin concentration. We have determined that quercetin inhibits formation of DNA supercoiling by gyrase from E. coli (Fig. 1B). In a separate experiment supercoiled DNA was used as a substrate for gyrase relaxation assay. Addition of quercetin promoted cleavage of DNA in the presence of gyrase at quercetin concentration above 80 μM (Fig. 1A) and maximal fraction of cleaved DNA is achieved at 640 μM. As a

Discussion

Inhibitory activity of flavonoids on class II topoisomerases has been demonstrated previously [21], [22]. It had been realized that the mechanism of inhibition is complex, however, it was generally assumed that the main binding target of flavonoids in topoisomerase II catalyzed reaction is DNA. Binding of flavonoids leads to the stabilization of the DNA–topo II complex causing cleavage of DNA [23]. Interaction of quercetin with single and double-stranded DNA has been shown by spectroscopic

Acknowledgements

This work was supported by the Ministry of Education, Science and Sport of the Republic of Slovenia and by KRKA d.d. pharmaceutical company. We thank Stanislav Zalar for his encouragement and support of the project and to Robert Bremšak for his excellent technical help.

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