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Structure-guided discovery of the metabolite carboxy-SAM that modulates tRNA function

Abstract

The identification of novel metabolites and the characterization of their biological functions are major challenges in biology. X-ray crystallography can reveal unanticipated ligands that persist through purification and crystallization. These adventitious protein–ligand complexes provide insights into new activities, pathways and regulatory mechanisms. We describe a new metabolite, carboxy-S-adenosyl-l-methionine (Cx-SAM), its biosynthetic pathway and its role in transfer RNA modification. The structure of CmoA, a member of the SAM-dependent methyltransferase superfamily, revealed a ligand consistent with Cx-SAM in the catalytic site. Mechanistic analyses showed an unprecedented role for prephenate as the carboxyl donor and the involvement of a unique ylide intermediate as the carboxyl acceptor in the CmoA-mediated conversion of SAM to Cx-SAM. A second member of the SAM-dependent methyltransferase superfamily, CmoB, recognizes Cx-SAM and acts as a carboxymethyltransferase to convert 5-hydroxyuridine into 5-oxyacetyl uridine at the wobble position of multiple tRNAs in Gram-negative bacteria1, resulting in expanded codon-recognition properties2,3. CmoA and CmoB represent the first documented synthase and transferase for Cx-SAM. These findings reveal new functional diversity in the SAM-dependent methyltransferase superfamily and expand the metabolic and biological contributions of SAM-based biochemistry. These discoveries highlight the value of structural genomics approaches in identifying ligands within the context of their physiologically relevant macromolecular binding partners, and in revealing their functions.

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Figure 1: Proposed chemical mechanism for the biosynthesis of cmo5U.
Figure 2: Structure of the CmoA–Cx-SAM complex.
Figure 3: Identification of low-molecular-weight compounds associated with CmoA-mediated Cx-SAM production.
Figure 4: In vitro assay of CmoB-catalysed carboxymethyltransfer activity.

Accession codes

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structure are deposited in the Protein Data Bank under the accession code 4GEK.

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Acknowledgements

We thank J. Parker and C. T. Walsh for providing the Aerobacter aerogenes 62-1 strain. We are indebted to V. Schramm and J. Gerlt for critical discussions and reading of the manuscript. This work was supported by US National Institutes of Health grants GM094662 (to S.C.A.), GM093342 (to S.C.A., M.P.J. and P.C.B.) and the Albert Einstein Cancer Center. This publication was made possible by the Center for Synchrotron Biosciences grant P30-EB-009998 from the National Institute of Biomedical Imaging and Bioengineering (NIBIB). Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract no. DE-AC02-98CH10886.

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J.K. carried out cloning, protein purification, crystallography, and functional assays. H.X. performed mass-spectrometry analysis of the in vitro assay. Y.-S.L. carried out LC–MS analysis of the CmoA-bound ligand and chemical synthesis of Cx-SAM. X.T. performed the NMR experiments. N.F.A.-O. carried out thermal denaturation studies. C.K. and M.P.J. performed computational modelling. S.B. and P.C.B. carried out the bioinformatics analysis. J.B.B. and Y.P. assisted in crystallographic validation and analysed crystallographic ligand-binding results. J.K. and S.C.A. designed the study, analysed the data and wrote the manuscript.

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Correspondence to Jungwook Kim or Steven C. Almo.

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Kim, J., Xiao, H., Bonanno, J. et al. Structure-guided discovery of the metabolite carboxy-SAM that modulates tRNA function. Nature 498, 123–126 (2013). https://doi.org/10.1038/nature12180

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