| dc.description.abstract | Tuberculosis, caused by Mycobacterium tuberculosis, is the leading cause of death due to infectious disease. Now, the prevalence of multidrug-resistant and extensively drug-resistant TB, and the emergence of co-infection of TB and HIV have highlighted the need for new antibiotics with novel mechanisms of action. Methionine aminopeptidase (MetAP) is a ubiquitous enzyme found in both prokaryotic and eukaryotic cells and carries out an important cotranslational modification of newly synthesized proteins. The MetAPs can be divided into type I and type II based on the existence of an insert in the catalytic domain. Prokaryotic cells have only one type of MetAP, either type I or type II; encoded by a single gene. MetAP is essential for cell viability, which is demonstrated by gene deletion experiment in E.coli and Salmonella typhimurium. Therefore, MetAP is a promising target for developing novel drugs against bacterial infection, including TB-causing drug resistance bacteria. Two genes, mapA and mapB , were found in Mycobacterium tuberculosis H37Rv. They encode two type I MetAP enzymes, MtMetAP Ia and MtMetAP Ic, respectively. Both MtMetAP proteins were over-expressed and purified in homogeneity as apoenzyme. Biochemical characterization using a fluorogenic substrate (Met-AMC) was carried out with both MtMetAP Ia and MtMetAP Ic. Both MtMetAPs can be activated by divalent metals, including Ni(II), Co(II), Mn(II) and Fe(II). Ni(II) is the best activator for both MtMetAPs, followed by Co(II) . Mn(II) and Fe(II) are the least efficient to activate MtMetAP Ia and MtMetAP Ic, respectively. Metal titration assays were used to determine the metal binding affinity to each MtMetAP. In both MtMetAP Ia and MtMetAP Ic, Co(II) and Fe(II) are the tightest binding metals, as indicated by their smallest Kd values. Mn(II) gives the weakest binding in MtMetAP Ia and Ni(II) shows a weakest binding to MtMetAP Ic. Growth complementation experiments were employed to evaluate the cellular function of MtMetAP in the E. coli that had an amber mutation in the chromosomal EcMetAP gene, and a pBAD plasmid, which encoded a suppressor tRNA to suppress the lethal effect of the amber mutation. The existence of glucose or arabinose in the culture medium could suppress or express the tRNA respectively, therefore result the death or survival of the E. coli, respectively. The plasmid-expressed MtMetAP Ic in the amber mutant rescued the E coli from death and supported cell growth. A set of inhibitors with selectivity for different metalated MetAPs were tested on both MtMetAPs. For MtMetAP Ib, all tested compounds retained their inhibitory activities and metal selectivity. However, in MtMetAP Ia, the Co(II)-, Mn(II)- and Fe(II)-selective inhibitors did not show inhibition. Only Fe(II)-selective inhibitors retained their inhibition, whereas they lost their metal selectivity. An amino acid sequence alignment suggested some differences in the active sites between MtMetAP 1a and MtMetAP Ic. A homology model of MtMetAP Ia based on MtMetAP Ic structure was generated. A similar active site is observed in this virtual structure of MtMetAP Ia. Given the size of the tested compound library, the failure to find an inhibitor specific for MtMetAP Ia may be due to the limited number of compounds in the library. Screening of a compound library consisting of a larger number of molecules with more structural diversity will possibly identify inhibitors for MtMetAP Ia. The inhibitors of MtMetAP Ic were further tested for their inhibition on cellular growth. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP Ic activity and inhibited the MtMetAP Ic-complemented cell growth, suggested that Fe(II) was the native metal used by MtMetAP1c in an E. coli cellular environment. X-ray structures of MtMetAP Ic in complex with three metalloform-selective inhibitors were analyzed. The results demonstrated different binding modes and different interactions with metal ions and active site residues for these inhibitors. The MtMetAP1c inhibitors with metalloform selectivity are potential leads for antitubercular drugs. Understanding the catalytic mechanism and inhibition of the mycobacterial MetAP is an essential step towards discovering and developing effective MetAP inhibitors as therapeutics. The compounds with potent inhibition and high metal selectivity toward MtMetAP may be therapeutically useful for improved TB treatment. | |
