The bacterial enzyme t-RNA (m1G37) methyltransferase (TrmD) is an ideal anti-microbial drug target since it is found in all eubacteria, serves an essential role during protein synthesis, and shares very little sequence or structural homology with its eukaryotic counterpart, Trm5. TrmD, a homodimeric protein, methylates the G37 nucleotide of tRNA using S-adenosyl-L-methionine (SAM) as the methyl donor and thus enables proper codon-anticodon alignment during translation. The two deeply buried binding sites for SAM seen in X-ray crystallography suggest that significant conformational changes must occur for substrate binding and catalytic turnover. Results from molecular dynamics simulations implicate a flexible loop region and a halo-like loop which may be gating the entrance to the active site. Analysis of simulation trajectories indicates an alternating pattern of active site accessibility between the two SAM binding sites, suggesting a single site mechanism for enzyme activity. Isothermal titration calorimetry (ITC), demonstrates that binding of SAM to TrmD is an exothermic reaction resulting from sequential binding at two sites. A similar mode of binding at higher affinities was observed for the product, S-adenosyl-L-homocysteine (SAH) suggesting that product inhibition may be important in vivo. ITC reveals that tRNA binding is an endothermic reaction in which one tRNA molecule binds to one TrmD dimer. This further supports the hypothesis of a single site mechanism for enzyme function. However, mutational analysis using hybrid mutant proteins suggests that catalytic integrity must be maintained in both active sites for maximum enzymatic efficiency. Mutations impeding flexibility of the halo loop were particularly detrimental to enzyme activity. Noncompetitive inhibition of TrmD was observed in the presence of bis-ANS, an extrinsic fluorescent dye. In silico ligand docking of bis-ANS to TrmD suggests that dye interferes with mobility of the flexible linker above the active site. Because SAM is a ubiquitous cofactor in methyltransferase reactions, analogs of this ligand may not be suitable for drug development. It is therefore important to investigate allosteric modes of inhibition. These experiments have identified key, mobile structural elements in the TrmD enzyme important for activity, and provide a basis for further research in the development of allosteric inhibitors for this enzyme.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-3724 |
| Date | 14 February 2012 |
| Creators | Palesis, Maria Kiouppis |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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