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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.

SUBMITTER: Holliday GL

PROVIDER: S-EPMC6445391 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Holliday Gemma L GL Akiva Eyal E Meng Elaine C EC Brown Shoshana D SD Calhoun Sara S Pieper Ursula U Sali Andrej A Booker Squire J SJ Babbitt Patricia C PC

Methods in enzymology 20180724

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 t ...[more]

PMID: 30097089

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Project description:RimO, encoded by the yliG gene in Escherichia coli, has been recently identified in vivo as the enzyme responsible for the attachment of a methylthio group on the beta-carbon of Asp88 of the small ribosomal protein S12 [Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 1826-1831]. To date, it is the only enzyme known to catalyze methylthiolation of a protein substrate; the four other naturally occurring methylthio modifications have been observed on tRNA. All members of the methylthiotransferase (MTTase) family, to which RimO belongs, have been shown to contain the canonical CxxxCxxC motif in their primary structures that is typical of the radical S-adenosylmethionine (SAM) family of proteins. MiaB, the only characterized MTTase, and the enzyme experimentally shown to be responsible for methylthiolation of N(6)-isopentenyladenosine of tRNA in E. coli and Thermotoga maritima, has been demonstrated to harbor two distinct [4Fe-4S] clusters. Herein, we report in vitro biochemical and spectroscopic characterization of RimO. We show by analytical and spectroscopic methods that RimO, overproduced in E. coli in the presence of iron-sulfur cluster biosynthesis proteins from Azotobacter vinelandii, contains one [4Fe-4S](2+) cluster. Reconstitution of this form of RimO (RimO(rcn)) with (57)Fe and sodium sulfide results in a protein that contains two [4Fe-4S](2+) clusters, similar to MiaB. We also show by mass spectrometry that RimO(rcn) catalyzes the attachment of a methylthio group to a peptide substrate analogue that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. Kinetic analysis of this reaction shows that the activity of RimO(rcn) in the presence of the substrate analogue does not support a complete turnover. We discuss the possible requirement for an assembled ribosome for fully active RimO in vitro. Our findings are consistent with those of other enzymes that catalyze sulfur insertion, such as biotin synthase, lipoyl synthase, and MiaB.

Dataset Information

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

Publications

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

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