One third of the world’s population is affected by Tuberculosis (TB), a disease caused by infection with Mycobacterium tuberculosis (MtB). The emergence of multidrug-resistant MtB makes this disease a major public health concern. New agents are needed to treat TB infections in a manner that circumvents existing pathways of resistance. One strategy is to target the organism at the translational level by inhibiting vital modifications of RNA. One gene responsible for these modifications is the tRNA (guanosine-1)-methyltransferase, trmD, which has been shown to be essential in several bacteria. The eukaryotic and bacterial m1G methyltransferases are structurally dissimilar, making this enzyme an ideal target for selective anti-TB agents. One strategy for TrmD inhibitor design is to target the catalytic center of the enzyme. Existing inhibitors such as Sinefungin exhibit poor selectivity due to the substrate’s role, SAM, as a universal methyl donor in many biological processes. Structure/activity relationships for inhibitory compounds are sparse, impeding the design of novel antimicrobials. Crystallographic data would identify molecular features unique to TrmD, and allow design of agents complimentary to the TrmD active site with minimal differential toxicity. Presently, no crystal structure for Mycobacterium tuberculosis TrmD exists. As a first step in this direction, the MtB gene has been cloned and expressed by using a His-tagged T7 expression vector. The recombinant protein was characterized through kinetic and preliminary inhibitor assays. The native enzyme displays a mass of 50 kDa, proving this enzyme is a dimer of two identical subunits. This is similar to data found on other TrmD orthologs. Crystallization of MtB TrmD has been achieved and preliminary x-ray diffraction studies conducted.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-3218 |
| Date | 29 July 2010 |
| Creators | Hamidi, Zohal |
| 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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