Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification.
Bottom Line: 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.Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM.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.
Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.Show MeSH
Mentions: While the determinants of cmo5U function are well defined, clarity has only recently begun to emerge regarding the relevant biosynthetic transformations leading to the cmo5U modification. Chorismate, or a related metabolite, had been implicated in the biosynthesis of cmo5U in E. coli, although the precise role of this compound in cmo5U formation was not clear (12). Subsequent mutagenesis experiments in S. enterica identified two members of the SDMT superfamily, CmoA and CmoB, as important components of the cmo5U biosynthetic pathway. cmoA-deficient mutants accumulate 5-hydroxyuridine (ho5U) and 5-methoxyuridine (mo5U), while cmoB mutants exclusively accumulate ho5U (13). The observation of mo5U was taken as evidence of its role as an obligate intermediate preceding cmo5U formation, which confounded the mechanistic interpretation of the cmo5U biosynthetic pathway. Recently, we demonstrated that cmo5U modification requires the previously unknown metabolite carboxy-S-adenosine-L-methionine (Cx-SAM), which is generated by the CmoA-catalyzed conversion of prephenate and SAM to yield Cx-SAM and phenylpyruvate. This remarkable reaction proceeds via a unique SAM-based sulfur-ylide intermediate, which captures the carbon dioxide moiety liberated as the consequence of decarboxylation of prephenate (14). Cx-SAM is subsequently utilized in the CmoB-catalyzed carboxymethylation of ho5U-containing tRNAs to yield the mature cmo5U-modified tRNAs (14), with no involvement of a mo5U intermediate (Figure 1). This transformation is notable as Cx-SAM must compete with SAM, which is one of the most abundant cofactors in nature (i.e. 180 μM in E. coli) (15–17). Hydroxylation of the wobble uridine is catalyzed by an as-yet-unidentified enzyme.
Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.