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

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

Managing Hazardous Municipal Wastewater: A Membrane-Integrated Hybrid Approach for Fast and Effective Treatment in Low Temperature Environment

Protection of natural water resources like lakes from the onslaught of hazardous municipal wastewater is often a challenge particularly in the cold regions. For treatment of enormous quantity of municipal wastewater, biological treatment is normally adopted but high COD (Chemical Oxygen demand) of such wastewater turns biological treatment slow and difficult. At low temperature environment, effective treatment of such municipal wastewater becomes extremely difficult due to weakened microbial activities. The present study was carried out with a hybrid approach comprising chemical treatment and membrane separation under psychrophilic conditions. Well–known Fenton’s treatment was adopted under response surface optimized conditions that helped recovery of nitrogen and phosphorus nutrients as value–added struvite fertilizer or magnesium ammonium phosphate (NH4MgPO4∙6H2O). The optimal COD removal was found to be 96% at a low temperature of 15oC and pH of 6.3 using Fe2+/H2O2 ratio of 0.10 and of H2O2 1.9 g/l with reaction time of 2 h. Down–stream purification of the struvite-free water by microfiltration and nanofiltration largely fouling–free flat sheet cross flow membrane modules ultimately turned the treated water reusable through reduction of dissolved solids, conductivity and salinity

A STUDY OF ORGANIZATIONAL CULTURE IN MANUFACTURING INDUSTRIES IN THE ROYAL KINGDOM OF BHUTAN

Research Objectives. The researchers have put following major objectives in the study, which contribute to Industrial Relations, Human Resource Management and overall performance of the manufacturing industries in Bhutan. 1. To study Organizational Culture (OC) in manufacturing industries in Bhutan. 2. To do comparative analysis of OC in manufacturing industries of different ownership in Bhutan. Research Results. Study of OC show that “Human Relations” culture in the companies is at high level though “Rational Goal” culture and “Internal Process” culture needs to be changed for better because the way how organizations do things has never changed very much. They do not have cultivated properly “Open Systems” culture. The comparative analysis of OC in three manufacturing industries of different ownership shows that “Human Relations”, “Rational Goal”, “Internal Process” cultures are better cultivated in the private company than in both of the government owned company and the Joint venture. Practical Significance of the Research. The practical significance of the work lies in the fact that the results of the research can be used to reorient the Industrial Relations and Human Resource Management practices in Bhutan towards an innovative path of development. Innovative ideas, practices and recommendations in the study can be used to improve the Organizational Culture , Organizational Behavior, Industrial Relations, which will lead to better performance of the company in terms of higher efficiency and effectiveness on the one hand and on the other hand higher social and emotional well-being of employees, which are the part of national goal of GNH in Bhutan. The results of the research are of interest to Industrial Relations’ specialists, lawyers, as well as for management people, who are engaged in collective agreement practices and social regulation of labor Relations. Research materials can be used in the process of teaching Personnel Management, Industrial Relations, Organizational behavior and other applied disciplines.Research Objectives. The researchers have put following major objectives in the study, which contribute to Industrial Relations, Human Resource Management and overall performance of the manufacturing industries in Bhutan. 1. To study Organizational Culture (OC) in manufacturing industries in Bhutan. 2. To do comparative analysis of OC in manufacturing industries of different ownership in Bhutan. Research Results. Study of OC show that “Human Relations” culture in the companies is at high level though “Rational Goal” culture and  “Internal Process” culture needs to be changed for better because  the way how organizations do things has never changed very much. They do not have cultivated properly “Open Systems” culture. The comparative analysis of OC in three manufacturing industries of different ownership shows that  “Human Relations”, “Rational Goal”, “InternalProcess” cultures  are better cultivated  in the private company than in both of the government owned company and the Joint venture. Practical Significance of the Research. The practical significance of the work lies in the fact that the results of the research can be used to reorient the Industrial Relations and Human Resource Management practices in Bhutan towards an innovative path of development. Innovative ideas, practices and recommendations in the study can be used to improve the Organizational Culture , Organizational Behavior, Industrial Relations, which will lead to better performance of the company in terms of higher efficiency and effectiveness on the one hand and on the other hand higher social and emotional well-being of  employees, which are the part of national goal of GNH in Bhutan. The results of the  research are of interest to Industrial Relations’ specialists, lawyers, as well as for management people, who are engaged in collective agreement practices and social regulation of labor Relations. Research materials can be used in the process of teaching Personnel Management, Industrial Relations, Organizational behavior and other applied disciplines

Seasonal variations in Home Range Size of Capped Langur (Trachypithecus pileatus) in a degraded habitat in Assam, India

A group of capped langurs, Trachypithecus pileatus comprising eight individuals was studied in Sri Surya Pahar, a degraded habitat in Goalpara district of Assam to record the seasonal variations in distance travelled, home range, and habitat utilization through direct observation supplemented by Geographical Information system (GIS). Scan sampling method was followed to record data on ranging behaviour. Seasonality in the home range size was evident and significant (P<0.01), it was 20 ha in winter, 17 ha in pre-monsoon, 17.75 ha in monsoon and 16.25 ha in retreating monsoon. The mean daily travel distance varied significantly (P<0.01), it was 375 m in retreating monsoon to 490 m in winter. The mean daily travel length was 439 m and the total annual home range size was 38.25 ha. The variation of home range size was correlated with the distribution and abundance of food resources. Home range size and daily travel distance showed significant seasonal variations. In both the cases the ranging patterns were longer during the winter season. This may be due to shortage of new leaves during winter, which is the preferred food item of capped langur. Spatial availability of the different food resources over different seasons may also be a reason for significant changes in ranging pattern during different seasons. The present data on home range size and ranging pattern of capped langur in degraded habitat could be useful for improvement of habitat and the conservation of this endangered species in Assam

REACTIVE OXYGEN SPECIES AS POSSIBLE MEDIATOR OF ANTIBACTERIAL ACTIVITY OF PARKIA JAVANICA, AGAINST BACTERIAL SPECIES PREDOMINANTLY FOUND IN CHRONIC WOUND.

The crude methanol extract of Parkia javanica was screened for antibacterial activity. against bacterial species predominantly found in chronic wound, by serial dilution technique. Growth kinetics study was performed and percentage of ROS production was measured by NBT reduction assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were obtained with a range of IC100 5-40 mg/ml in case of standard bacterial strains. The lag phase of all extract treated bacteria is extended compared to untreated cells. The normalized % of ROS is increased in presence of crude extract. This study suggests that the crude methanol extract of Parkia javanica possesses promising antimicrobial substances which are having activity against Standard ATCC bacterial species and ROS induced DNA damage could be the possible mediator of its antimicrobial activity. Keywords: Parkia javanica, antibacterial activity, standard ATCC bacterial strains, growth curve, ROS, DNA damag

Lewis acid-induced rearrangment of α-[bis(methylthio)methylene]ethyl-2-styrylcyclopropylcarbinols: unexpected formation of a novel bicyclo[3.2.1]octadiene framework

The α-[bis(methylthio)methylene]ethyl-2-styrylcyclopropylcarbinols 9a-c undergo a simple but unexpected skeletal rearrangement in the presence of stannic chloride in nitromethane to afford bicyclo[3.2.1]octadiene derivatives 10a-c exclusively in good yields. The structure of 10a was conclusively elucidated by X-ray diffraction studies. A possible mechanism governing the formation of 10 is proposed

Pinpointing The Extent Of Electronic Delocalization In The Re(i)-to-tetrazine Charge-separated Excited State Using Time-resolved Infrared Spectroscopy

Femtosecond mid-IR transient absorption spectroscopy (TRIR) and time-dependent density functional theory (TD-DFT) calculations on Re(CO)(3)Cl(Me(2)BPTZ) [Me(2)BPTZ = 3,6-bis(5-methyl-2-pyridine)-1,2,4,5-tetrazine] are used to demonstrate that the lowest excited state of the complex is a triplet metal-to-ligand charge-transfer ((3)MLCT) state with a lifetime of 225 ps. The short excited-state lifetime is explained by the energy-gap taw. Vibrational cooling of the (3)MLCT state shows up as early-time dynamics (3.6 ps). The structural changes in the excited state are deduced from the frequency shifts in the TRIR vibrational bands. The vibrational frequencies of the CO groups increase upon excitation as a result of decreased back-bonding between the CO ligands and the oxidized Re center in the (3)MLCT state. The vibrational frequencies of the central tetrazine ring of Me(2)BPTZ decrease because of the decrease in the bond order upon reduction of the Me(2)BPTZ ligand in the (3)MLCT state. Interestingly, the TRIR signals from the pyridine moieties of Me2BPTZ were not detected. These results can be explained by localization of the electronic charge on the central tetrazine ring in the (3)MLCT state of Re(CO)(3)Cl(Me(2)BPTZ), as supported by TD-DFT calculations

Research for practice in small human service organisations: doing and disseminating smallscale research

A series of novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one (DHPM) derivatives were designed, synthesized and evaluated in vitro as potential inhibitors of chorismate mutase (CM). All these compounds were prepared via a multi-component reaction (MCR) involving sequential I2-mediated Biginelli reaction followed by Cu-free Sonogashira coupling. Some of them showed promising inhibitory activities when tested at 30 μM. One compound showed dose dependent inhibition of CM with IC50 value of 14.76 ± 0.54 μM indicating o-alkynylphenyl substituted DHPM as a new scaffold for the discovery of promising inhibitors of CM

Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5'-terminal regions of cap0-, cap1- and 5'ppp- mRNAs.

Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA's 5'-terminal 'cap'. The minimal 'cap0' consists of N7-methylguanosine linked to the first nucleotide via a 5'-5' triphosphate (ppp) bridge. Cap0 is further modified by 2'-O-methylation of the next two riboses, yielding 'cap1' (m7GpppNmN) and 'cap2' (m7GpppNmNm). However, some viral RNAs lack 2'-O-methylation, whereas others contain only ppp- at their 5'-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5'ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2'-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5'-terminal regions of 5'ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5'-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5'ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations