Limits...
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.

Show MeSH
Overall structure of CmoB. (A) g(s*) distribution is fit with data from sedimentation velocity experiments for a single-species model. The best value for S20,w is 7.27S, with an expected molecular weight of 154.28 kD. The calculated molecular weight of a monomer based on the amino acid sequences is 37.01 kD. (B) A tetramer of Cx-SAM bound CmoB, where the N-terminal 100 residues unique to CmoB are highlighted in blue. Cx-SAM and phosphate ions are displayed in sticks, where carbon atoms are in gray, oxygen atoms in red, nitrogen atoms in blue, sulfur atoms in yellow and phosphorus atoms in orange. (C) A monomer of Cx-SAM bound CmoB and (D) apo-CmoB.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482062&req=5

Figure 2: Overall structure of CmoB. (A) g(s*) distribution is fit with data from sedimentation velocity experiments for a single-species model. The best value for S20,w is 7.27S, with an expected molecular weight of 154.28 kD. The calculated molecular weight of a monomer based on the amino acid sequences is 37.01 kD. (B) A tetramer of Cx-SAM bound CmoB, where the N-terminal 100 residues unique to CmoB are highlighted in blue. Cx-SAM and phosphate ions are displayed in sticks, where carbon atoms are in gray, oxygen atoms in red, nitrogen atoms in blue, sulfur atoms in yellow and phosphorus atoms in orange. (C) A monomer of Cx-SAM bound CmoB and (D) apo-CmoB.

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.


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)

Overall structure of CmoB. (A) g(s*) distribution is fit with data from sedimentation velocity experiments for a single-species model. The best value for S20,w is 7.27S, with an expected molecular weight of 154.28 kD. The calculated molecular weight of a monomer based on the amino acid sequences is 37.01 kD. (B) A tetramer of Cx-SAM bound CmoB, where the N-terminal 100 residues unique to CmoB are highlighted in blue. Cx-SAM and phosphate ions are displayed in sticks, where carbon atoms are in gray, oxygen atoms in red, nitrogen atoms in blue, sulfur atoms in yellow and phosphorus atoms in orange. (C) A monomer of Cx-SAM bound CmoB and (D) apo-CmoB.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Overall structure of CmoB. (A) g(s*) distribution is fit with data from sedimentation velocity experiments for a single-species model. The best value for S20,w is 7.27S, with an expected molecular weight of 154.28 kD. The calculated molecular weight of a monomer based on the amino acid sequences is 37.01 kD. (B) A tetramer of Cx-SAM bound CmoB, where the N-terminal 100 residues unique to CmoB are highlighted in blue. Cx-SAM and phosphate ions are displayed in sticks, where carbon atoms are in gray, oxygen atoms in red, nitrogen atoms in blue, sulfur atoms in yellow and phosphorus atoms in orange. (C) A monomer of Cx-SAM bound CmoB and (D) apo-CmoB.
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.

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