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Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification.

Kim J, Xiao H, Koh J, Wang Y, Bonanno JB, Thomas K, Babbitt PC, Brown S, Lee YS, Almo SC - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

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Cx-SAM binding site of CmoB. (A) Fo-Fc difference Fourier synthesis, calculated at 2.6-Å resolution without the ligand, contoured at 3σ around the modeled Cx-SAM ligand. (B) Polar interactions between Cx-SAM and CmoB. A cross-eye stereo presentation of Cx-SAM bound in the CmoB catalytic site, where amino acid residues within 3.5 Å of the ligand are shown. Hydrogen bonding and ionic interactions are depicted in dashed lines. (C) A cross-eye stereo presentation of an overlay of active sites of Cx-SAM bound and apo CmoB structures. Liganded CmoB residues are colored in light pink, and residues from the apo-structure are in green. An identical color scheme is applied to Cx-SAM and sulfate ion as in Figure 2.
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Figure 3: Cx-SAM binding site of CmoB. (A) Fo-Fc difference Fourier synthesis, calculated at 2.6-Å resolution without the ligand, contoured at 3σ around the modeled Cx-SAM ligand. (B) Polar interactions between Cx-SAM and CmoB. A cross-eye stereo presentation of Cx-SAM bound in the CmoB catalytic site, where amino acid residues within 3.5 Å of the ligand are shown. Hydrogen bonding and ionic interactions are depicted in dashed lines. (C) A cross-eye stereo presentation of an overlay of active sites of Cx-SAM bound and apo CmoB structures. Liganded CmoB residues are colored in light pink, and residues from the apo-structure are in green. An identical color scheme is applied to Cx-SAM and sulfate ion as in Figure 2.

Mentions: Difference Fourier syntheses calculated after initial rounds of refinement, without the inclusion of ligand, exhibited features consistent with the binding of Cx-SAM in the catalytic site (Figure 3A). Continued refinement and modeling indicated that Cx-SAM is present at full occupancy and demonstrated similar atomic interactions in all 10 independent catalytic sites in both liganded structures.


Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification.

Kim J, Xiao H, Koh J, Wang Y, Bonanno JB, Thomas K, Babbitt PC, Brown S, Lee YS, Almo SC - Nucleic Acids Res. (2015)

Cx-SAM binding site of CmoB. (A) Fo-Fc difference Fourier synthesis, calculated at 2.6-Å resolution without the ligand, contoured at 3σ around the modeled Cx-SAM ligand. (B) Polar interactions between Cx-SAM and CmoB. A cross-eye stereo presentation of Cx-SAM bound in the CmoB catalytic site, where amino acid residues within 3.5 Å of the ligand are shown. Hydrogen bonding and ionic interactions are depicted in dashed lines. (C) A cross-eye stereo presentation of an overlay of active sites of Cx-SAM bound and apo CmoB structures. Liganded CmoB residues are colored in light pink, and residues from the apo-structure are in green. An identical color scheme is applied to Cx-SAM and sulfate ion as in Figure 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482062&req=5

Figure 3: Cx-SAM binding site of CmoB. (A) Fo-Fc difference Fourier synthesis, calculated at 2.6-Å resolution without the ligand, contoured at 3σ around the modeled Cx-SAM ligand. (B) Polar interactions between Cx-SAM and CmoB. A cross-eye stereo presentation of Cx-SAM bound in the CmoB catalytic site, where amino acid residues within 3.5 Å of the ligand are shown. Hydrogen bonding and ionic interactions are depicted in dashed lines. (C) A cross-eye stereo presentation of an overlay of active sites of Cx-SAM bound and apo CmoB structures. Liganded CmoB residues are colored in light pink, and residues from the apo-structure are in green. An identical color scheme is applied to Cx-SAM and sulfate ion as in Figure 2.
Mentions: Difference Fourier syntheses calculated after initial rounds of refinement, without the inclusion of ligand, exhibited features consistent with the binding of Cx-SAM in the catalytic site (Figure 3A). Continued refinement and modeling indicated that Cx-SAM is present at full occupancy and demonstrated similar atomic interactions in all 10 independent catalytic sites in both liganded structures.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Show MeSH
Related in: MedlinePlus