Molecular genetic and biochemical analysis of thymidylate synthase and dihydrofolate reductase from mycobacteria

Abstract

Mycobacterial diseases, including tuberculosis, leprosy and desseminated Mycobacterium avium disease, continue to ravage mankind with high levels of morbidity and mortality worldwide. The development of novel strategies to control these diseases is essential. The dissertation was aimed at characterization of mycobarterial thymidylate synthase (TS) and dihydrofolate reductase (DHFR). These enzymes are components of the dTMP synthesis cycle, a key biosynthetic pathway that provides for de novo synthesis of dTMP, a precursor of DNA. The genes encoding TS (thyA) and DHFR (folA) have been cloned from M. avium, BCG, M. leprae, M. smegmatis and M. tuberculosis by a combination of complementation of a defined E. coli thyA mutant with a BCG expression library, and cross-hybridization of the resulting clone with cosmid libraries of the above mentioned species of mycobacteria. In all cases the folA gene was located immediately downstream of thyA. The DNA sequences of thyA and folA from BCG and M. tuberculosis were found to be identical. Primer extension analysis has been used to map the 5{dollar}\sp\prime{dollar} end of the mRNA for BCG thyA and the close proximity of folA to thyA suggests that they may be in a single operon. The DNA sequences for M. avium, M. leprae and M. smegmatis thyA and folA have been determined and found to show significant homologies.;In order to characterize the biochemical properties of these enzymes from M. tuberculosis, heterologous expression systems were employed. In the case of TS, a T7 based expression system was used to achieve an expression level of about 5-7% of total soluble protein. The expressed protein was purified to apparent homogeneity by sequential application of ion exchange chromatography, hydrophobic interaction chromatography and dye affinity chromatography. Kinetic analysis of the purified protein indicated that the Km for the substrate was significantly higher than it is in other TS's. A pH rate profile analysis suggests that an active site histidine may play a role in catalysis.;In the case of DHFR, a construct selected directly from the BCG expression library was used. The expressed protein was purified to apparent homogeneity by sequential application of methotrexate-affinity cromatography, followed by ion exchange chromatography. Kinetic analysis of the purified protein yielded information about the Km for the substrate and cofactor, the pH optima for the reaction and the effects of salts and urea on the rate of reaction. It was later determined from sequence analysis and hybridization that the origin of the construct is not BCG but is likely to be some other mycobacteria based on phylogenetic analysis.