The role of aspartic acid 143 in E. coli tRNA-guanine transglycosylase: Insights from mutagenesis studies and computational modeling
Katherine A. Todorov 1, Xiao-Jian Tan 2, Susanne T. Nonekowski 3, George A. Garcia 2 and Heather A. Carlson 2*
1 Armed Forces Institute of Pathology
2 University of Michigan, Ann Arbor
3 The University of Toledo
* To whom correspondence should be addressed. E-mail: carlsonh{at}umich.edu.
Submitted on January 12, 2005
Revised on February 22, 2005
Accepted on 27 May 2005
 |
Abstract |
|---|
tRNA guanine transglycosylase (TGT) is a tRNA-modifying enzyme which catalyzes the posttranscriptional exchange of guanine in position 34 of tRNA(Y,H,N,D) with the modified base queuine in eukaryotes or its precursor, preQ1 base, in eubacteria. Thus, TGT must recognize the guanine in tRNA and the free base queuine or preQ1 to catalyze this exchange. The crystal structure of Z. mobilis TGT with preQ1 bound suggests that a key aspartate is critically involved in substrate recognition. To explore this, a series of site-directed mutants of D143 in E. coli TGT were made and characterized to investigate heterocyclic substrate recognition. Our data confirm that D143 has significant impact on KM of guanine; however, the trend in the KM data (D143A < D143N < D143S < D143T) is unexpected. Computational studies were used to further elucidate the interactions between guanine and the D143 mutants. A homology model of E. coli TGT was created and the role of D143 was investigated by molecular dynamic simulations of guanine bound to the wild-type and D143-mutant TGTs. To validate the model systems against our kinetic data, free energies of binding were fit using the Linear Interaction Energy (LIE) method. This is a unique application of the LIE method because the same ligand is bound to several mutant proteins rather than one protein binding several ligands. The atomic detail gained from the simulations provided a better understanding of the binding affinities of guanine with the mutant TGTs, revealing that water molecules enter the active site, hydrogen bond to the ligand, and compensate for lost protein-ligand interactions. The trend of binding affinity for wt > D143A > D143N > D143S > D143T appears to be directly related to the degree of hydrogen bonding available to guanine in the binding site.
Key Words:
Linear Interaction Energy (LIE), Molecular Dynamics, RNA modification, TGT, bridging water, heterocyclic recognition
