Polyphosphate Kinase from M. tuberculosis: An Interconnect between the Genetic and Biochemical Role

PLOS ONE, Dec 2010

The enzyme Polyphosphate Kinase (PPK) catalyses the reversible transfer of the terminal γ-Pi of ATP to form a long chain Polyphosphate (PolyP). Using an IPTG inducible mycobacterial vector, the vulnerability of this gene has been evaluated by antisense knockdown experiments in M. tuberculosis. Expression profiling studies point to the fact that down regulation of PPK caused cidality during the late phase in contrast to its bacteriostatic mode immediately following antisense expression. PPK thus seems to be a suitable anti-tubercular drug target. The enzyme which is a tetramer has been cloned in E. coli and purified to homogeneity. An enzyme assay suitable for High Throughput Screening was optimized by using the statistical Taguchi protocol and the kinetic parameters determined. The enzyme displayed a strong product inhibition by ADP. In order to accurately estimate the product inhibition, progress curve analysis of the enzyme reaction was monitored. The kinetic equation describing the progress curve was suitably modified by taking into account the product inhibition. The reversible nature of the enzyme indicated a possibility of a two way ATP↔ADP switch operating in the bacteria as a response to its growth requirement.

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Polyphosphate Kinase from M. tuberculosis: An Interconnect between the Genetic and Biochemical Role

Datta S (2010) Polyphosphate Kinase from M. tuberculosis: An Interconnect between the Genetic and Biochemical Role. PLoS ONE 5(12): e14336. doi:10.1371/journal.pone.0014336 Polyphosphate Kinase from M. tuberculosis : An Interconnect between the Genetic and Biochemical Role Vijayalakshmi Jagannathan 0 Parvinder Kaur 0 Santanu Datta 0 David M. Ojcius, University of California Merced, United States of America 0 AstraZeneca India Pvt. Ltd. , Bangalore , India The enzyme Polyphosphate Kinase (PPK) catalyses the reversible transfer of the terminal c-Pi of ATP to form a long chain Polyphosphate (PolyP). Using an IPTG inducible mycobacterial vector, the vulnerability of this gene has been evaluated by antisense knockdown experiments in M. tuberculosis. Expression profiling studies point to the fact that down regulation of PPK caused cidality during the late phase in contrast to its bacteriostatic mode immediately following antisense expression. PPK thus seems to be a suitable anti-tubercular drug target. The enzyme which is a tetramer has been cloned in E. coli and purified to homogeneity. An enzyme assay suitable for High Throughput Screening was optimized by using the statistical Taguchi protocol and the kinetic parameters determined. The enzyme displayed a strong product inhibition by ADP. In order to accurately estimate the product inhibition, progress curve analysis of the enzyme reaction was monitored. The kinetic equation describing the progress curve was suitably modified by taking into account the product inhibition. The reversible nature of the enzyme indicated a possibility of a two way ATPADP switch operating in the bacteria as a response to its growth requirement. - Inorganic Polyphosphate (PolyP), a likely precursor in pre-biotic evolution, is a ubiquitous entity that is found in diverse locations such as volcanic condensates, deep oceanic steam vents and inside living cells. They are linear polymers in the size range of 31000 of orthophosphate residues linked by high-energy phospho-anhydride bonds. Though this polymer was identified more than a century ago, its biochemical role has only been clarified in the last decade through the pioneering work by Kornberg and colleagues [14]. In nature, polyphosphate is formed by dehydration of orthophosphate at elevated temperature, while its cellular synthesis is catalyzed by the enzyme Polyphosphate Kinase (PPK) which uses the c-Pi of ATP to extend the polymer. Its reverse reaction is the formation of ATP from ADP and Pi [2]. Although this polymer is found in nearly all types of bacteria, its level fluctuates orders of magnitude depending on the physiologic and metabolic state of the cell. Its cellular accumulation is in response to specific physiological states - like deficiencies in amino acid, Pi, nitrogen or to the more general stresses akin to a nutrient downshift or high salt. It seems that the intracellular polyphosphate level is maintained by shifting the equilibrium between the forward and the reverse rate of the enzyme reaction. An interesting observation has been the stage specific essentiality of the gene. Mutants of E. coli (ppk2) have severe growth defects in the stationary phase while showing normal growth kinetics in the logarithmic zone. Stationary phase of growth as studied in the laboratory can be thought of resembling the stressful and deprived state that characterizes the natural habitat of most bacteria. To cope with such adverse conditions, stationary phase cells undergo drastic physiological and morphological changes and a number of genes are induced in this phase in order that the cells survive [5]. Localization of PolyP granules in the vicinity of bacterial nucleoid suggests their possible involvement in regulation of gene activity, which may be essential for adaptation to stationary phase and other stresses [6]. Thus, not only polyphosphate could act as an essential marker for stress response, but it also might supply the organism of the much needed ATP to cope during its nutrient downshift or environmental stress. Additionally, the phosphoanhydride bonds can be used as a high energy source for phosphorylation of glucose as its free energy of hydrolysis is almost equal to that of ATP. PPK has also been implicated in virulence and in-vivo growth in several bacteria. In view of the phylogenetic similarity of the enteropathogens and the frequency with which virulence factors are expressed in stationary phase, mutants lacking the ppk gene in pathogenic Shigella flexneri, Salmonella enterica serovar dublin, and Salmonella enterica serovar typhimurium showed decreased virulence phenotype such as: (i) growth defects, (ii) defective responses to stress and starvation, (iii) loss of viability, (iv) polymyxin sensitivity, (v) intolerance to acid and heat, and (vi) diminished invasiveness in epithelial cells. A ppk mutant of Pseudomonas aeruginosa was shown to be defective in quorum sensing and the dependent virulence factors, elastase (...truncated)


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Vijayalakshmi Jagannathan, Parvinder Kaur, Santanu Datta. Polyphosphate Kinase from M. tuberculosis: An Interconnect between the Genetic and Biochemical Role, PLOS ONE, 2010, 12, DOI: 10.1371/journal.pone.0014336