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.
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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)