Abstract
DNA gyrase is a bacterial DNA topoisomerase that catalyzes ATP-dependent negative DNA supercoiling and DNA decatenation. The enzyme is a heterotetramer comprising two GyrA and two GyrB subunits. Its overall architecture is conserved, but species-specific elements in the two subunits are thought to optimize subunit interaction and enzyme function. Toward understanding the roles of these different elements, we compared the activities of Bacillus subtilis, Escherichia coli, and Mycobacterium tuberculosis gyrases and of heterologous enzymes reconstituted from subunits of two different species. We show that B. subtilis and E. coli gyrases are proficient DNA-stimulated ATPases and efficiently supercoil and decatenate DNA. In contrast, M. tuberculosis gyrase hydrolyzes ATP only slowly and is a poor supercoiling enzyme and decatenase. The heterologous enzymes are generally less active than their homologous counterparts. The only exception is a gyrase reconstituted from mycobacterial GyrA and B. subtilis GyrB, which exceeds the activity of M. tuberculosis gyrase and reaches the activity of the B. subtilis gyrase, indicating that the activities of enzymes containing mycobacterial GyrB are limited by ATP hydrolysis. The activity pattern of heterologous gyrases is in agreement with structural features present: B. subtilis gyrase is a minimal enzyme, and its subunits can functionally interact with subunits from other bacteria. In contrast, the specific insertions in E. coli and mycobacterial gyrase subunits appear to prevent efficient functional interactions with heterologous subunits. Understanding the molecular details of gyrase adaptations to the specific physiological requirements of the respective organism might aid in the development of species-specific gyrase inhibitors.
Highlights
DNA gyrase is a bacterial DNA topoisomerase that catalyzes ATP-dependent negative DNA supercoiling and DNA decatenation
To investigate the functional interaction of subunits from B. subtilis, E. coli, and M. tuberculosis, we analyzed the supercoiling activity of homologous gyrases, assembled from GyrA and GyrB from the same organism, and heterologous gyrases harboring GyrA and GyrB from different organisms
In contrast to B. subtilis and E. coli gyrase, mycobacterial gyrase is a very inefficient enzyme that shows slow, DNA-independent hydrolysis of ATP and low supercoiling and decatenation activities
Summary
DNA gyrase is a bacterial DNA topoisomerase that catalyzes ATP-dependent negative DNA supercoiling and DNA decatenation. Toward understanding the roles of these different elements, we compared the activities of Bacillus subtilis, Escherichia coli, and Mycobacterium tuberculosis gyrases and of heterologous enzymes reconstituted from subunits of two different species. DNA topology is regulated by enzymes of the topoisomerase family (reviewed in Ref. 4), which catalyze the relaxation or introduction of negative and positive supercoils or resolve catenanes (reviewed in Ref. 5). Type II topoisomerases bind covalently to the 5Ј-ends of the cleaved DNA [6] They are divided into type IIA and IIB subfamilies according to structural similarities [11]. The type IA topoisomerase reverse gyrase and the type IIA topoisomerase gyrase catalyze the introduction of positive or negative supercoils, respectively. The introduction of DNA supercoils is energetically disfavored, and both enzymes couple these reactions to ATP hydrolysis Type IB enzymes catalyze relaxation in an ATPindependent reaction through controlled rotation of the DNA [15,16,17], whereas type II topoisomerases relax DNA in an ATPdependent reaction through a strand-passage mechanism (reviewed in Ref. 18)
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