Functional Analyses of the Toxoplasma gondii DNA Gyrase Holoenzyme: A Janus Topoisomerase with Supercoiling and Decatenation Abilities.

Ting-Yu Lin,Soshichiro Nagano,Jonathan Gardiner Heddle

doi:10.1038/srep14491

Ting-Yu Lin, Soshichiro Nagano + Show 1 more

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https://doi.org/10.1038/srep14491

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Abstract

A number of important protozoan parasites including those responsible for toxoplasmosis and malaria belong to the phylum Apicomplexa and are characterised by their possession of a relict plastid, the apicoplast. Being required for survival, apicoplasts are potentially useful drug targets and their attractiveness is increased by the fact that they contain “bacterial” gyrase, a well-established antibacterial drug target. We have cloned and purified the gyrase proteins from the apicoplast of Toxoplasma gondii (the cause of toxoplasmosis), reconstituted the functional enzyme and succeeded in characterising it. We discovered that the enzyme is inhibited by known gyrase inhibitors and that, as well as the expected supercoiling activity, it is also able to decatenate DNA with high efficiency. This unusual dual functionality may be related to the apparent lack of topoisomerase IV in the apicoplast.

Highlights

As a result of its bacterial origin and the possession of a circular genome, the apicoplast retains a “bacterial” DNA gyrase protein (“gyrase”)[12]
Mycobacterium tuberculosis is an unusual example of a species of bacteria lacking topo IV and it has been found that, in order to compensate for this absence, its gyrase (MtGyr) has substantial functional differences compared to gyrase from E. coli (EcGyr)
Given some apparent similarities between MtGyr and TgGyr including the fact that both appear to exist in environments without topo IV, we examined the effect of calcium on TgGyr activities by performing supercoiling assays in the absence of either magnesium or calcium (Fig. 5a) and found that replacing magnesium ions with calcium ions abolished supercoiling activity

Summary

Introduction

As a result of its bacterial origin and the possession of a circular genome, the apicoplast retains a “bacterial” DNA gyrase protein (“gyrase”)[12]. Mycobacterium tuberculosis is an unusual example of a species of bacteria lacking topo IV and it has been found that, in order to compensate for this absence, its gyrase (MtGyr) has substantial functional differences compared to gyrase from E. coli (EcGyr) It has enhanced DNA relaxation and decatenation activities[19]. (fluoro)quinolones appear to be ineffective in treating apicomplexan diseases in vivo with ciprofloxacin and norfloxacin having been tested[27,28,29] This may be due to difficulty in accessing the interior of the apicoplast, which requires the traversal of at least seven membranes, or it may be due to structural/functional differences in the apicoplast gyrases themselves, making them resistant to the drugs. Full length P. falciparum GyrA has proved resistant to attempts to produce and purify it, meaning that comprehensive biochemical testing of the functional enzyme has not been achieved

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