Functional interactions between gyrase subunits are optimized in a species-specific manner

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.

Towards Conformation-Sensitive Inhibition of Gyrase: Implications of Mechanistic Insight for the Identification and Improvement of Inhibitors

“…Although gyrases share the common type IIA topoisomerase scaffold, enzymes from different bacteria contain species-specific elements [ 72 , 73 , 74 ]. A comparison of gyrases from Escherichia coli , B. subtilis , and M. tuberculosis reveals that the B. subtilis enzyme is a minimal version.…”

“…Gyrase activities are further modulated by sequence and structural variations in the GyrA CTDs [ 13 , 75 , 76 , 77 , 78 ]. As a result, enzymes from different bacteria have different supercoiling set-points, and a different balance between wrapping-dependent supercoiling and wrapping-independent decatenation of DNA [ 74 , 75 ]. This species-specific modulation of gyrase activity and the potential variations in the underlying mechanisms have important ramifications for inhibitor identification and structure-guided drug design: on one hand, these differences can be exploited to identify compounds that target the species-specific insertion, offering the possibility to develop highly effective gyrase inhibitors specific for a particular enzyme.…”

“…( B ) The species-specific insertion elements modulate the supercoiling activity: B. subtilis and E. coli gyrase are efficient supercoiling enzymes that supercoil half of the DNA in less than 30 s. In contrast, M. tuberculosis gyrase catalyzes supercoiling more slowly, with a half-life of 2–5 min. The supercoiling endpoints also differ [ 74 ]. ( C ) The insertion elements also modulate the decatenation activity of the different gyrases.…”

“…M. tuberculosis gyrase is also the slowest enzyme in decatenation, with a half-life of >15 min. Figure modified from [ 74 ].…”

Diversity and Functions of Type II Topoisomerases

“…Уникальная особенность ДНК-гиразы -способность вносить отрицательную суперспирализацию с затратой энергии гидролиза АТР, что показано в экспериментах in vitro для ферментов E. coli, B. subtilis, C. crescentus, M. tuberculosis и многих других бактерий. Кроме того, ДНК-гираза эффективно релаксирует положительные супервитки и способна декатенировать кольцевые молекулы ДНК [39,[53][54][55][56].…”