Structural Perspectives on the Mechanism of the Radical SAM Enzyme, Spore Photoproduct Lyase

Abstract

Spore Photoproduct Lyase (SP lyase) is a unique DNA repair enzyme, conferring extreme UV-resistance to bacterial spores and hence, contributing to the resistance mechanism of several pathogenic species among Bacilli and Clostridia classes. SP lyase catalyzes the repair of a UV-induced thymidine dimer, however, unlike DNA photolyases, it belongs to the superfamily of radical SAM enzymes and involves a unique but still ill-defined DNA repair mechanism [1]. Recently, we succeeded to solve the first crystal structure of SP lyase, allowing us to describe at the molecular level, the interactions between SP lyase, the SAM co-factor, the [4Fe-4S] center and its DNA lesion substrate called “spore photoproduct” [2]. The structures pointed out several amino acid residues likely critical for the enzyme mechanism. Further structural and functional investigations lead us to identify a possible H-atom transfer (HAT) pathway in the enzyme active site. To probe this mechanistic hypothesis, we rationally rewired the enzyme HAT pathway in a SP lyase defective mutant. For the first time, we demonstrated that it is possible to engineer a radical SAM enzyme active site, supporting our mechanistic hypothesis and opening new avenues for the development of novel radical biocatalysts. Finally, our study enables us to draw unanticipated structural and mechanistic relationships among radical SAM enzymes, enlightening how these enzymes can control radical chemistry within their active sites.