<b>A unified view on enzyme catalysis by cryo-EM study of a DNA topoisomerase</b> - Supplementary Data files

Substrate recognition is crucial in enzyme catalysis. However, the theories that elucidate the process still require further inspection. Here we use cryogenic electron microscopy (cryo-EM) and African swine fever virus type 2 topoisomerase (AsfvTop2) to demonstrate the substrate binding theories in an ordered manner: catalytic selection by the enzyme, conformational selection by the substrates, and then induced-fit. The apo-AsfvTop2 co-exists in six conformers that comply with the two-gate mechanism directing DNA passage and release in the Top2 catalytic cycle. The structures of AsfvTop2-DNA-inhibitor complexes show that, besides several unique features, substantial induced-fit changes occur locally from the closed apo-conformer that however is too far-fetched for the open apo-conformer. Furthermore, the ATPase domain of AsfvTop2 in the MgAMP-PNP-bound crystal structures coexist in reduced and oxidized forms involving a disulfide bond, which can regulate the AsfvTop2 function, a mechanism unique to topoisomerases and a likely evolutionary strategy for viral survival. These findings unravel the structural features of AsfvTop2 that are applicable in antiviral development, and also we propose that the three-stage mechanism for the initiation of enzyme catalysis is likely universal, though not always all observable.