Atlas of the Radical SAM Superfamily: Divergent Evolution of Function Using a “Plug and Play” Domain

Abstract

The radical SAM superfamily contains over 100,000 homologous enzymes that catalyze a remarkably broad range of reactions required for life, including metabolism, nucleic acid modification, and biogenesis of cofactors. While the highly conserved SAM-binding motif responsible for formation of the key 5'-deoxyadenosyl radical intermediate is a key structural feature that simplifies identification of superfamily members, our understanding of their structure-function relationships is complicated by the modular nature of their structures, which exhibit varied and complex domain architectures. To gain new insight about these relationships, we classified the entire set of sequences into similarity-based subgroups that could be visualized using sequence similarity networks. This superfamily-wide analysis reveals important features that had not previously been appreciated from studies focused on one or a few members. Functional information mapped to the networks indicates which members have been experimentally or structurally characterized, their known reaction types, and their phylogenetic distribution. Despite the biological importance of radical SAM chemistry, the vast majority of superfamily members have never been experimentally characterized in any way, suggesting that many new reactions remain to be discovered. In addition to 20 subgroups with at least one known function, we identified additional subgroups made up entirely of sequences of unknown function. Importantly, our results indicate that even general reaction types fail to track well with our sequence similarity-based subgroupings, raising major challenges for function prediction for currently identified and new members that continue to be discovered. Interactive similarity networks and other data from this analysis are available from the Structure-Function Linkage Database.

Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens

“…S2), the conversion of q to preQ 1 , a reaction never described previously to our knowledge, is needed to complete the pathway. The third gene of the CD1682 operon encodes for a protein of the radical-SAM (RS) enzyme family, known for its remarkable and versatile enzymology (47)(48)(49)(50). We hypothesized that CD1684 (UniProt ID Q186P0) could act as a lyase, breaking a C-N bond to generate preQ 1 from q.…”

Mechanism of activation of the human cysteine desulfurase complex by frataxin

“…Fe-S clusters are protein cofactors that are necessary for many critical biochemical processes (9,10). Fe-S clusters have a variety of enzymatic roles, including electron transfer, substrate binding and activation, and the initiation of radical chemistry (11,12). Different Fe-S cluster stoichiometries, such as the [2Fe-2S] and [4Fe-4S] forms, are synthesized and distributed to apo target proteins by conserved biosynthetic pathways.…”

Accumulation of an unprecedented 5′-deoxyadenos-4′-yl radical unmasks the kinetics of the radical-mediated C-C bond formation step in MoaA catalysis

“…If this cluster is in the reduced form during the catalytic cycle, it may then be responsible for the reduction of the putative aminyl radical intermediate. The redox roles have been proposed for auxiliary clusters in the radical SAM enzymes in the SPASM/Twitch family27,55,60 , and the redox potentials have been reported for enzymes under resting states61 . However, no experimental demonstration has been made about their redox states during the catalytic cycle.…”