Recent progress in radical SAM enzymes: New reactions and mechanisms

Abstract

<p indent="0mm"><italic>S</italic>-adenosyl-<italic>L</italic>-methionine (SAM) is the second largest cofactor in the human body, and its metabolism is closely related to various physiological activities. In addition to a methyl donor, SAM is also used by radical SAM enzymes with a [4Fe-4S] cluster to catalyze a series of radical reactions. Since the discovery of this enzyme family, it has been found to play important physiological functions in all kingdoms of life. According to bioinformatics prediction, over 220000 radical SAM enzymes are involved in more than 85 biochemical transformations. Almost all radical SAM enzymes have a conserved CxxxCxxC motif, which coordinates the [4Fe-4S] cluster. The reduced [4Fe-4S] cluster provides an electron to SAM to cleave the C_{5′,adenosine}−S bond of SAM and generates a 5′-deoxyadenosine radical (5′-dA·). This radical then grabs a hydrogen atom from the substrate to generate the substrate radical and initiates many different types of reactions. These include thioether crosslinking reactions, radical addition reactions that generate C–C bonds, aliphatic etherification that generates C−O bonds, oxidation reactions, complex rearrangement reactions, methylation, methylthiolation, cyclopropanation reactions and so on. The nonclassical radical SAM enzyme Dph2 cleaves the C_γ,Met–S bond of SAM to generate a 3-amino-3-carboxylpropyl radical (ACP radical), which is added to a histidine residue of the substrate protein elongation factor EF2 for diphthamide biosynthesis. Radical SAM enzymes have a very rich substrate scope, including ribosomally synthesized and posttranslationally modified peptides (RiPPs), proteins, nucleosides and all kinds of small molecules. Classifying by the type of substrate, this review summarizes some of the newly discovered radical SAM enzymes since 2015 and introduces the catalytic mechanism. In addition to new enzymes and reactions, strides have been made in mechanistic studies of radical SAM enzymes in the past several years. Using rapid freeze-quench (RFQ) combined with electron spin resonance spectroscopy (EPR) and electron-nucleus double resonance spectroscopy (ENDOR), researchers have captured and characterized two types of novel intermediates in radical SAM enzymes. These two intermediates are organometallic species with Fe–C bonds formed by the iron-sulfur cluster with the 5′-dA radical and ACP radical, respectively. These findings answer the long-standing question of how enzymes control active organic radical species in radical SAM enzymes and the regioselectivity of SAM cleavage. Finally, we look forward to the future directions of radical SAM enzymes. As discussed above, radical SAM enzymes catalyze numerous difficult reactions, such as C−H activation and C−C bond formation. New enzymatic reactions with unnatural substrates could be developed with protein engineering. Therefore, natural product analogs could be easily made for drug development. Furthermore, in the radical SAM chemistry, only Dph2 can cleave SAM unconventionally to generate the ACP radical, other than the 5′-dA radical from all the other radical SAM enzymes. Whether there are more enzymes using this mechanism to catalyze the reaction remains unexplored. We expect that more Dph2-like enzymes that can generate ACP radicals will be discovered in the future. Finally, the drawback for almost all the reported radical SAM enzymes thus far is their low efficiency. Protein engineering or other strategies that could increase the stability and efficiency of radical SAM enzymes are in high demand. Only by this means would we expect more widespread use of radical SAM enzymes as biocatalysts in synthetic biology.