One of our foci is the functions and mechanisms of a group of enzymes called radical S-adenosylmethionine (SAM) enzymes, one of the largest groups of enzymes with > 113,000 functional domains. These enzymes catalyze the reductive cleavage of S-adenosyl-L-methionine (SAM) to generate transient 5’-deoxyadenosyl radical (5′-dA•), which is subsequently used to catalyze a variety of free radical-mediated reactions (see Figure below). Many of these enzymes are found in biological processes closely associated with human diseases. Also, these enzymes catalyze free radical-mediated reactions, which until recently, were considered rare in enzyme catalysis. Thus, their functions and mechanisms form the foundation of a novel paradigm in enzymology. In this project, our interests are two folds; (1) discovery of novel reactions by radical SAM enzymes and (2) mechanisms of radical initiation and control by radical SAM enzymes.
(1) Functional characterization of radical SAM enzymes.
Among radical SAM enzymes, one of our current foci is C-C bond forming reactions essential for the construction of the carbon backbone of cofactors and natural products. Because C-C bond formations are frequently the key steps in metabolic pathways, characterizations of enzymes responsible for such reactions are critical for the understanding of the pathways. So far, we have successfully discovered novel reactions catalyzed by two enzymes; MoaA in molybdenum cofactor biosynthesis (JACS 2013; PNAS 2015) and NikJ/PolH in antifungal nucleoside biosynthesis (Nat. Chem. Biol. 2016). As detailed below, each of these findings has provided breakthrough understanding in these biosynthetic pathways.
(2) Mechanism of radical initiation and control by radical SAM enzymes
We are also studying the fundamental mechanisms of radical SAM enzyme catalysis, such as radical initiation and control. The critical questions for this part of the project are:
• How do radical SAM enzymes generate 5′-dA•? What is the structural trigger for the radical initiation? (JACS 2015)
• How do radical SAM enzymes control the reactivities of highly reactive intermediates (JACS 2020)?
These questions are common to all radical SAM enzymes, and therefore addressing them will improve our understanding of radical SAM enzymes in general. To this end, we take multidisciplinary approaches including, small molecule probes, EPR, ENDOR, X-ray crystallography, NMR, and electrochemistry.