NMR-based structural analysis of threonylcarbamoyl-AMP synthase and its substrate interactions.
 

NMR-based structural analysis of threonylcarbamoyl-AMP synthase and its substrate interactions.

Related Articles NMR-based structural analysis of threonylcarbamoyl-AMP synthase and its substrate interactions.

J Biol Chem. 2015 Jun 9;

Authors: Harris KA, Bobay BG, Sarachan KL, Sims AF, Bilbille Y, Deutsch C, Iwata-Reuyl D, Agris PF

Abstract
The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon-codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP (TC-AMP), a key intermediate in the biosynthesis of t(6)A37, the details of the enzymatic mechanism remain unsolved. Therefore, the solution structure of Escherichia coli TsaC was characterized by NMR in order to further study the interactions with ATP and L-threonine, both substrates of TsaC in the biosynthesis of TC-AMP. Several conserved amino acids were identified that create a hydrophobic binding pocket for the adenine of ATP. Additionally, two residues were found to interact with L-threonine. Both binding sites are located in a deep cavity at the center of the protein. Models derived from the NMR data and molecular modeling reveal several sites with considerable conformational flexibility in TsaC that may be important for L-threonine recognition, ATP activation and/or protein-protein interactions. These observations further the understanding of the enzymatic reaction catalyzed by TsaC, a threonylcarbamoyl-AMP synthase, and provide structure-based insight into the mechanism of t(6)A37 biosynthesis.


PMID: 26060251 [PubMed - as supplied by publisher]



More...