Although the tremendous catalytic power of enzymes is widely recognized, their exact mechanisms of action are still a source of debate. In order to elucidate the origin of their power, it is necessary to look at individual residues and atoms, and establish their contribution to ligand binding, activation, and reaction. Given the present limitations of experimental techniques, only computational tools allow for such detailed analysis. During my PhD studies I have applied a variety of computational methods, reviewed in Chapter 2, to the study of two enzymes: DfrB dihydrofolate reductase (DHFR) and methyltetrahydrofolate: corrinoid/iron-sulfur protein methyltransferase (MeTr).
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The DfrB enzyme has intrigued microbiologists since it was discovered thirty years ago, because of its simple structure, enzymatic inefficiency, and its insensitivity to trimethoprim. This bacterial enzyme shows neither structural nor sequence similarity with its chromosomal counterpart, despite both catalysing the reduction of dihydrofolate (DHF) using NADPH as a cofactor. As numerous attempts to obtain experimental structures of an enzyme ternary complex have been unsuccessful, I combined docking studies and molecular dynamics simulations to produce a reliable model of the reactive DfrBDHFNADPH complex. These results, combined with published empirical data, showed that multiple binding modes of the ligands are possible within DfrB.
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Comprehensive sequence and structural analysis provided further insight into the DfrB family. The presence of the dfrB genes within integrons and their level of sequence conservation suggest that they are old structures that had been diverging well before the introduction of trimethoprim. Each monomer of the tetrameric active enzyme presents an SH3-fold domain; this is a eukaryotic auxiliary domain never found before as the sole domain of a protein, let alone as the catalytic one. Overall, DfrB DHFR seems to be a poorly adapted catalyst, a minimalistic enzyme that promotes the reaction by facilitating the approach of the ligands rather than by using specific catalytic residues.
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MeTr initiates the Wood-Ljungdahl pathway of anaerobic CO2 fixation. It catalyses the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH3THF) to the cobalt centre of a corrinoid/iron-sulfur protein. For the reaction to occur, the N5 position of CH3THF is expected to be activated by protonation. As experimental studies have led to conflicting suggestions, computational approaches were used to address the activation mechanism.
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Initially, I tested the accuracy of quantum mechanical (QM) methods to predict protonation positions and pKas of pterin, folate, and their analogues. Then, different protonation states of CH3THF and active-site aspartic residues were analysed. Fragment QM calculations suggested that the pKa of N5 in CH3THF is likely to increase upon protein binding. Further, ONIOM calculations which accounted for the complete protein structure indicated that active-site aspartic residues are likely to be protonated before the ligand. Finally, solvation and binding free energies of several protonated forms of CH3THF were compared using the thermodynamic integration approach. Taken together, these preliminary results suggest that further work with particular emphasis on the protonation state of active-site aspartic residues is needed in order to elucidate the protonation and activation mechanism of CH3THF within MeTr.
| Identifer | oai:union.ndltd.org:ADTP/216874 |
| Date | January 2006 |
| Creators | Alonso, Hernan, hernan.alonso@anu.edu.au |
| Publisher | The Australian National University. The John Curtin School of Medical Research |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.anu.edu.au/legal/copyrit.html), Copyright Hernan Alonso |
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