Tuberculosis emerged from its grave to be one of the deadliest diseases of the present time after recently developing a synergy with AIDS. A fatty acid condensing enzyme-mtFabH has been proposed to connect the key processes involved in biosynthesis of mycolic acids, an important component of mycobacterial cell wall. It condenses long acyl Coenzymes A (CoA; up to C20CoA) with malonyl Acyl Carrier Protein (ACP) to form the elongated β-ketoacyl-ACP which further undergoes rounds of elongation to form mero-mycolate branch of mature mycolic acids. Owing to its proposed central position in mycolic acid synthesis, mtFabH has attracted considerable attention as a good anti-mycobacterial target.In this study, we utilized important biochemical tools such as site directed mutagenesis, mass spectrometry and X-ray crystallography to address some of the key unanswered questions regarding the intricate workings of mtFabH. We solved the first co-crystal structure of substrate C12CoA with mtFabH and further analyzed the substrate specificity of this acylation step. This structure depicts the mode of acyl-CoA binding in mtFabH channels; and its comparison with the parallel E.Coli-acetyl CoA structure provides important similarities and differences in substrate binding in these two FabH enzymes. It also posed an important question about the trajectory of long acyl chain CoA into the deep and "seemingly closed" substrate binding pocket of mtFabH. By utilizing disulfide-based inhibitors, we showed that large conformational changes are necessary to facilitate ligand trafficking in mtFabH while the high catalytic turnover rate of the enzyme is maintained. We also proposed the most likely location of the involved loop.A much faster and less cumbersome assay for mtFabH was also developed and it was utilized to characterize a series of inhibitors. This assay utilizes the commercially available radioactive malonyl-CoA in lieu of malonyl-ACP, the physiological substrate, and thus can serve as ACP independent assay for mtFabH.These studies further our understanding of the biochemistry of mtFabH, which along with the faster assay could be helpful in designing potent mtFabH inhibitors as anti-tubercular agents in the future.

The main aim of the study in this thesis was to use structure-based protocols to design new drugs for enzymes, DXS and DXR in the non mevalonate pathway. Another aim of this study was to identify the dimer interface in E.coli FabH as an allosteric binding site for designing new class of anti-infective drugs. We have attempted to identify potential inhibitors for DXS by docking the NCI Diversity set compounds, compound libraries available from GSK-MMV and St. Jude's Children's research center. FabH dimer interface has been identified as a potential target using SiteMap, Alanine mutagenesis and docking studies. The first chapter gives an overview of the computational methods. The next two chapters briefly introduce the biological targets in the author's study. Chapter two explains the importance of non-mevalonate pathway in microbes. Different enzymes in the non-mevalonate pathway are discussed and the importance of terpenoids in biological processes and also the use of terpenoids as drugs have been extensively discussed in this chapter. The crystal structures available for DXS and DXR are also discussed. Chapter three brings out the importance of FabH as an anti-infective target. Crystal structure of FabH E.coli is discussed and the importance of FabH as a dimer has been discussed in this chapter. Chapter 3 describes the methods, homology models generated, and analysis from docking studies. The homology models for PvDXS and PvDXR have been used in this study to identify potential inhibitors. Domain swapping and the structural organization of PvDXS before and after domain swaping are discussed. Identification of domain swaping in PvDXS using entropy changes has been extensively discussed. Chapter 4 focuses on FabH (Fatty Acid Biosynthesis, enzyme H also referred to as β-ketoacyl-ACP-synthase III) dimer interface as an allosteric target. SiteMap analysis and MD simulations on the FabH monomer and dimer structures revealed the dimer interface as a binding region. Further analyses were done by mutagenesis studies on the Phe87 residue, a key residue at the dimer interface region and validating the results using docking studies. NCI Diversity Set compounds were docked at the dimer interface of FabH, which revealed that compounds NSC91529 and NSC19803 docked best at the dimer interface region with the phenyl ring of both the compounds

Work in the Turos group over the past five years has focused on the development of N-alkylthio beta-lactams, which show antibacterial activity against Staphylococcus (including MRSA), Bacillus, and others. These compounds do not function in the manner of the traditional beta-lactam antibiotics, but were thought to undergo an intracellular thiol-transfer to coenzyme A. In expanding the SAR of these novel compounds, it was found that N-alkylthio-2-oxazolidinones also exhibit antibacterial activity. Although CoA acts as the thiol-redox buffer in the genera most susceptible to the N-alkylthio beta-lactams, studies on Coenzyme A disulfide reductase (CoADR) show that the redox buffer is not affected by these compounds. However, the recent finding that fatty acid synthesis is affected by the N-alkylthio beta-lactams led to the discovery that these compounds act as prodrugs, and that the asymmetric CoA disulfides produced by in vivo thiol transfer are potent inhibitors of beta-ketoacyl-acyl carrier protein synthase III (FabH) through a novel thiol-disulfide exchange with the active site cysteine. Lactams 2a and 2g were also found to be potent inhibitors of this enzyme. In an effort to produce a CoA mixed-disulfide mimic which could cross the cell membrane, a series of simple aryl-alkyl disulfides were synthesized and tested against E. coli, S. aureus, and B. subtilis. Several of these compounds were found to be very potent antibacterials both in vitro and in vivo, with MICs less than 0.125 micrograms/mL. Comparison of the activities of these disulfides with those of acyl-CoA analogs and CoA mixed disulfides support the assertion that FabH is indeed the cellular target of these potent new compounds.