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: Sedimentation velocity analysis of CmoB is consistent with a tetramer that is insensitive to Cx-SAM or SAM (Figure 2A). Experimental phases derived from selenomethionyl-substituted CmoB crystals were used to determine all three structures described herein (Supplementary Table S1). Cx-SAM bound CmoB structures were determined in two crystal forms, resulting in 10 independent copies of the complex (two tetramers in one crystal form, and one dimer in the other), while the apo structure contains a single dimer in the asymmetric unit (Figure 2). The dimers exhibit symmetry-related interactions that recapitulate the expected tetramer. Pairwise structural alignments of all 12 protomers (calculated on 319–323 Cα's) displayed RMSDs ranging from 0.10 to 0.51 Å, with the most variable region being a short loop composed of Gln-35, His-36 and Gly-37. This segment, which is more than 15 Å from the catalytic site, is disordered in seven out of the 12 chains in the three structures.
Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.