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

Kim J, Xiao H, Bonanno JB, Kalyanaraman C, Brown S, Tang X, Al-Obaidi NF, Patskovsky Y, Babbitt PC, Jacobson MP, Lee YS, Almo SC. (2013) Nature 498, 123–6. PMCID: PMC3895326

In collaboration, the EFI and New York Structural Genomics Research Consortium (NYSGRC) discovered a novel metabolite, Cx-SAM, and defined its biosynthetic pathway.  NYSGRC identified Cx-SAM as a ligand that unexpectedly persisted through purification, crystallization, and structure determination of an uncharacterized enzyme from the SAM-Dependent Methyltransferase (SDMT) Superfamily. Experimental follow-up coupled with input from the EFI Computation and Superfamily/Genome Cores revealed previously unknown activities involved in tRNA modification and defined greater functional diversity in the SDMT superfamily. This effort further highlights the strength of coupling multiple disciplines with superfamily-based analyses to expand our knowledge of metabolism and in particular represents an outstanding example of integration between large-scale consortia.

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. a, Previously identified biosynthetic pathway for cmo5U at wobble uridines. First, the wobble uridine is converted to ho5U by an unknown mechanism, and this is followed by the action of CmoA and CmoB. b, Mechanism for CmoA-catalysed Cx-SAM formation from SAM and prephenate. c, Mechanism for CmoB-catalysed formation of cmo5U from ho5U and Cx-SAM.

Figure 2. Proposed chemical mechanism for the biosynthesis of cmo5U. a, Previously identified biosynthetic pathway for cmo5U at wobble uridines. First, the wobble uridine is converted to ho5U by an unknown mechanism, and this is followed by the action of CmoA and CmoB. b, Mechanism for CmoA-catalysed Cx-SAM formation from SAM and prephenate. c, Mechanism for CmoB-catalysed formation of cmo5U from ho5U and Cx-SAM.

Figure 3. Identification of low-molecular-weight compounds associated with CmoA-mediated Cx-SAM production. a, Electrospray time-of-flight (ESI-TOF) mass spectra of a Cx-SAM standard (top) and the low-molecular-weight compound co-purifying with CmoA (bottom). Peak mass-to-charge ratio (m/z) were 443.1345 and 443.1349 (errors, −0.9 and +2.3 p.p.m.) for Cx-SAM standard and the compound that co-purified with recombinant CmoA, respectively. b, Detection of Cx-SAM in an in vitro assay containing SAM, chorismate and CmoA. c, Detection of phenylpyruvate (C9H7O3) formation in the assay mixture by mass spectrometry in negative mode (m/z = 163.0398 observed, 163.0395 calculated). d, Time course of Cx-SAM production in an in vitro assay of CmoA. The assay solution contained 20 mM sodium phosphate, pH 6.8, 0.2 mM [14C-methyl]-SAM, 0.2 mM prephenate or chorismate, and 2 μM CmoA. Error bars represent the s.d. of three data sets. e, Time course of the phenylpyruvate formation from prephenate. The assay mixture contained 20 mM sodium phosphate, pH 6.8, 0.2 mM prephenate, 0.2 mM SAM (open circles and inverted triangles) and 2 μM CmoA (open circles and filled circles). Error bars represent the s.d. of three data sets. f, Solvent proton exchange of [2H3-methyl]-SAM catalysed by CmoA. The sample contained 10 mM Tris, pH 8.0, 0.5 mM [2H3-methyl]-SAM and 10 μM CmoA, with or without 0.5 mM prephenate. The reaction was carried out at room temperature (20 to 25 °C) for 4 h. In the presence of prephenate, doubly deuterated SAM (calculated m/z = 401.1576) was observed. tR, retention time.

Figure 4. In vitro assay of CmoB-catalysed carboxymethyltransfer activity. Total RNA extracted from CmoB mutant cells was used as a substrate, and Cx-SAM was generated in situ by the action of CmoA on prephenate and SAM. RNA was digested with P1 nuclease to 5′-nucleotide monophosphates and then mass-spectrometry analysis was carried out. Left, no CmoB was added; right, addition of purified E. coli CmoB resulted in the detection of cmoUMP (C11H14O12N2P, m/z = 397.0282 observed, 397.0284 calculated) in negative ion mode.

Reprinted with permission from Nature Publishing Group. Copyright © 2013, Rights Managed by Nature Publishing Group