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: It was reported that total tRNA derived from cmoA-deficient mutants possessed both ho5U and mo5U, in contrast to wild-type total tRNA which possessed predominately cmo5U (13). The presence of small amounts of mo5U has also been reported previously from total RNA (23) and tRNA (24) of wild-type E. coli. When we examined P1 nuclease-treated total tRNA from wild-type E. coli by LC-MS/MS, a trace amount of mo5U was indeed detected in addition to the much more abundant cmo5U (Supplementary Figure S5). Enrichment of mo5U was observed in ΔcmoA- and ΔaroC-mutants, where aroC encodes the protein catalyzing the formation of chorismate, the immediate precursor of prephenate required for the CmoA-catalyzed transformation (Supplementary Figure S5). No significant amount of cmo5U was detected in these mutants, as these mutants are unable to synthesize Cx-SAM (12,13,25). Both mo5U and cmo5U were absent from ΔcmoB-mutant, in agreement with a previous report (13). These findings suggest that in addition to Cx-SAM-dependent cmo5U formation, CmoB catalyzes the SAM-dependent methylation of wobble ho5U in Gram-negative organisms, albeit with much lower efficiency. To investigate the in vivo generation of mo5U and cmo5U, modifications were monitored in cmoB-deficient cells complemented with a plasmid directing the inducible production of wild-type cmoB. Total RNA was extracted from cells at 3-, 6- and 24-h time points after the induction of CmoB expression, and nucleotides derived from P1 nuclease treatment were analyzed by LC-MS (Figure 4). Three hours post-induction of cmoB expression, cmo5U was detected in the sample, while no mo5U was observed. The MS peak corresponding to mo5U began to appear 6 h post-induction, confirming that CmoB is involved in the in vivo generation of both cmo5U and mo5U.
Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.