Insights Into The Mechanistic Details Of The M.Tuberculosis Pantothenate Kinase : The Key Regulatory Enzyme Of CoA Biosynthesis

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis, has long been the scourge of humanity, claiming millions of lives. It is the most devastating infectious disease of the world in terms of mortality as well as morbidity (WHO, 2009). The lack of a uniformly effective vaccine against TB, the development of resistance in the Mycobacterium tuberculosis against the present antitubercular drugs and its synergy with AIDS has made the situation very alarming. This therefore necessitates a search for new antitubercular drugs as well as the identification of new and unexplored drug targets (Broun et aI., 1992). Coenzyme A is an essential cofactor for all organisms and is synthesized in organisms from pantothenate by a universally conserved pathway (Spry et al., 2008; Sassetti and Rubin, 2003). The first enzyme of the pathway, pantothenate kinase catalyzes the most important step of the biosynthetic process, being the first committed step of CoA biosynthesis and the one at which all the regulation takes place (Gerdes et aI., 2002) This thesis describes the successful cloning of PanK from Mycobacterium tuberculosis, its expression in E. coli, single step affinity purification, and complete biochemical and biophysical characterization. In this work, pantothenol, a widely believed inhibitor of pantothenate kinase, has been shown to act as a substrate for the mycobacterial pantothenate kinase. Further it was shown that the product, 4'phosphopantothenol, thus formed, inhibited the next step of the CoA biosynthesis pathway in vitro. The study was extended to find outthe fate of pantothenol inside the cell and it was demonstrated that the CoA biosynthetic enzymes metabolized the latter into the pantothenol derivative of CoA which then gets incorporated into acyl carrier protein. Lastly, it was decisively shown that pantothenate kinase is not only regulated by feedback inhibition by CoA but, also regulated through feed forward stimulation by Fructose 1, 6 biphosphate (FBP), a glycolytic intermediate. The binding site of FBP was determined by docking and mutational studies of MtPanK. Chapter 1 presents a brief survey of the literature related to Coenzyme A biosynthesis pathway and describes the objective of the thesis. It also presents a history of TB and briefly reviews literature describing TB as well as the life cycle, biology, survival strategy, mode of infection and the metabolic pathways operational in the TB parasite, Mycobacterium tuberculosis. The chapter details the enzymes involved in CoA biosynthesis pathway from various organims. Chapter 2 In this chapter, cloning of the ORF (Rv1092c), annotated as pantothenate kinase in the Tuberculist database (http://genolist.pasteur.frfTubercuList), its expression in E. coli and purification using affinity chromatography has been described. Protein identity was confirmed by MALDI-TOF and by its ability to complement the pantothenate kinase temperature sensitive mutant, DV70. This chapter also illustrates the oligomeric status of MtPanK in solution and describes the biochemical characterization of MtPanK by means of two different methods, spectrophotometrically by a coupled assay and calorimetrically by using Isothermal Titration Calorimetry. Feedback inhibition of MtPanK by CoA is also discussed in this chapter. Chapter 3 describes the biophysical characterization of MtPanK. It discusses the enthalpy (~H) and free energy change (~G) accompanying the binding of a non-hydrolysable analogue of ATP; CoA; acetyl CoA and malonyl-CoA to MtPanK. The chapter details the energetics observed upon ATP binding to pantothenate-saturated MtPanK further elucidating the order of the reaction. This chapter also describes the various strategies which were designed and tested to remove CoA from the enzyme as the latter is always purified from the cell in conjunction with CoA. Validation of these strategies for complete CoA removal (by studying the n value from ITC studies) is further described. Chapter 4 discusses the interaction of the well-studied inhibitor of pantothenate kinases from other sources (e.g. the malarial parasite), pantothenol, with the mycobacterial enzyme. In order to investigate the interaction of this compound with MtPanK, its effect on the kinetic reaction carried out by the enzyme was studied by several methods. Surprisingly, a new band corresponding to 4'phosphopantothenol appeared when the reaction mix of MtPanK with pantothenol and ATP was separated on TLC. The identity of the new spot was confirmed by mass spectrometry analyses of the MtPanK reaction mixture.. These findings established the fact that pantothenol is a substrate of pantothenate kinase. To delve deeper into the mechanism of interaction of this compound with the enzymes of the coenzyme A biosynthesis pathway, the ability of pantothenol to serve as a substrate for the next step of the pathway, MtCoaBC was studied. Using various approaches it was established that pantothenol is actually a substrate for the MtPanK and the inhibition observed earlier (Saliba et aI., 2005) is actually due to the inability of CoaBC to utilize 4' -phosphopantothenol as substrate. Chapter 5 takes the story from Chapter 4 further detailing the effects of pantothenol on cultures of E. coli and M. smegmatis. I observed that pantothenol does not inhibit the culture of E. coli or M. smegmatis. So, further studies were carried out to know the fate of pantothenol once it is converted into 4'phosphopantothenoi. Since, the next enzyme of the pathway does not utilize 4'phosphopantothenol, I checked the further downstream enzyme of the pathway, CoaD, and found that it converts 4'-phosphopantothenol to thepantothenol derivative of dephospho-CoA. The next enzyme of the pathway, CoaE, took up this pantothenol derivative of dephospho-CoA as a substrate and converted it to the pantothenol derivative of CoA which was then transferred to apo-ACP by holo-ACP synthase. The holo-ACP thus synthesized enters into the dedicated pathway of fatty acid synthesis. Extensive investigations have been carried out on the regulation of pantothenate kinases, by the product of the pathway, Coenzyme A and its thioesters, xx establishing the latter as the feedback regulators of these enzymes. In order to determine if the cell employs mechanisms to sense available carbon sources and consequently modulate its coenzyme A levels by regulating activity of the enzymes involvedin CoA biosynthesis, glycolytic intermediates were tested against MtPanK for their possible role in the regulation of MtPanK activity. Chapter 6 details my identification of a novel regulator of MtPanK activity, fructose-I, 6-bisphosphate (FBP), a glycolytic intermediate, which enhances the MtPanK catalyzed phosphorylation of pantothenate by three fold. Further, the possible mechanisms through which FBP mediates MtPanK activation are also discussed. This chapter also describes the experiments carried out to identify the binding site of FBP on MtPariK.Interestingly, docking of FBP on MtPanK revealed that FBP binds close to the ATP binding site on the enzyme with one of its phosphates overlapping with the 3'~phosphate of CoA thereby validating its competitive binding relative to CoA on MtPanK. Based on these observations I propose that the binding of FBP to MtPanK lowers the activation energy of pantothenate phosphorylation by PanK. Chapter 7 presents a summary of the findings of this work. Coenzyme A biosynthesis pathway harbors immense potential in the development of drug against many communicable diseases, thanks to its essentiality for the pathogens and the differences between the pathogen and host CoA biosynthetic enzymes. The work done in this thesis extensively characterizes the first committed enzyme of the CoA biosynthetic pathway, pantothenate kinase, from Mycobacterium tuberculosis (MtPanK). The thesis also deals with the fate of a known inhibitor of PanK and proves it as a substrate for MtPanK. Finally this thesis describes a new link between glycolysis and CoA biosynthesis. Biotin, like coenzyme A, is another essential cofactor required by several enzymes in critical metabolic pathways. De novo synthesis of this critical metabolite has been reported only in plants and microorganisms. Therefore targeting the synthesis of biotin in the tubercular pathogen is another effective means of handicapping the tubercle pathogen. During the course of my studies, I also investigated the mycobacterial biotin biosynthesis pathway, studying the first enzyme of the pathway, 7-keto-8-aminopelargonic acid (KAPA) synthase (bioF) in extensive detail. Appendix 1 elucidates the kinetic properties of 7-keto-8aminopelargonic acid synthase (bioF) from Mycobacterium tuberculosis and proves beyond doubt that D-alanine which has previously been reported to act as a competitive inhibitor for the B. sphaericus enzyme (Ploux et al., 1999), is actually a substrate for the mycobacterial bioF.