Abstract
Enzyme-mediated modifications at the wobble position of tRNAs are essential for the translation of the genetic code. We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs. CmoB is distinctive in that it is the only known member of the SAM-dependent methyltransferase (SDMT) superfamily that utilizes a naturally occurring SAM analog as the alkyl donor to fulfill a biologically meaningful function. Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM. Complementary high-resolution structures of the apo- and Cx-SAM bound CmoB reveal the determinants responsible for this remarkable discrimination. Together, these studies provide mechanistic insight into the enzymatic and non-enzymatic feature of this alkyl transfer reaction which affords the broadened specificity required for tRNAs to recognize multiple synonymous codons.
Highlights
All organisms contain fewer than 61 unique tRNA species, precluding a direct one-to-one mapping between anticodons and sense codons
When we examined P1 nuclease-treated total tRNA from wild-type E. coli by liquid chromatography-tandem mass spectrometry (LC-MS)/MS, a trace amount of mo5U was detected in addition to the much more abundant cmo5U (Supplementary Figure S5)
Genetic and structural data demonstrate that the primary in vivo function of CmoB is as a carboxymethyl transferase in the biosynthesis of cmo5U-modified tRNAs
Summary
All organisms contain fewer than 61 unique tRNA species, precluding a direct one-to-one mapping between anticodons and sense codons This incongruence requires some anticodons to recognize multiple codons for complete translation of the genetic code. The needed degeneracy derives from non-canonical Watson–Crick pairings between the 3 nucleotide of a codon triplet and the ‘wobble’ nucleotide at the 5 -end of an anticodon [1] These wobble nucleotides are the targets for numerous enzyme-mediated modifications, which are important for fundamental aspects of translation, including the broadened specificity required for degenerate codon recognition [2,3,4], ribosome binding [5,6], reading frame maintenance [7] and translocation [8]. Direct crystallographic analysis revealed the structural and chemical features responsible for the expanded codon recognition properties exhibited by cmo5U [11], including an intramolecular hydrogen between the ether oxygen of the oxyacetyl moiety and the 2 -hydroxyl of U33, which promotes an anticodon stemloop conformation that supports degenerate binding to multiple codons
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