PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, Mycobacterium tuberculosis

Unité de Biochimie Structurale, URA 2185 CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France.
Molecular Microbiology (Impact Factor: 4.42). 10/2003; 49(6):1493-508. DOI: 10.1046/j.1365-2958.2003.03657.x
Source: PubMed

ABSTRACT Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles. Here we focus on the catalytic domains of two trans-membrane enzymes, the Ser/Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis. PstP was found to specifically dephosphorylate model phospho-Ser/Thr substrates in a Mn2+-dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP-mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phosphorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth.

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    • "The M. tuberculosis genome also includes three genes (pstP, ptpA, and ptpB) encoding eukaryotic-like protein phosphatases in addition to STPKs. PstP is a metalloenzyme that belongs to the PPM family of Ser/Thr protein phosphatases, which is known to dephosphorylate various mycobacterial STPKs and their substrates (Boitel et al., 2003; Chopra et al., 2003; Durán et al., 2005). "
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    ABSTRACT: Tuberculosis continues to be a major cause of mortality worldwide despite significant advances in chemotherapy and development of the BCG vaccine. Although curable, the tuberculosis treatment period (6-9 months) presents many concerns, including patient noncompliance and the development of drug toxicity and drug resistance. This study aimed to understand the protein-protein interactions of key proteins involved in the Mycobacterium tuberculosis STPK signal transduction pathway (such as PknB, PknE, and PstP); in addition, we attempted to identify promising leads for the inhibition of protein-protein interactions. Interactome analyses revealed the interactions of these protein targets with several other proteins, including PknG and PbpA. Drug-like candidates were screened based on Lipinski's rule of five and the absorption digestion metabolism excretion toxicity. Molecular docking of the target proteins with the selected ligands identified cryptolepine HCl to be a common molecule interacting with all protein targets (with a good docking score). The generation of a pharmacophore model for cryptolepine HCl revealed three pharmacophoric regions: aromatic hydrocarbon, hydrogen bond acceptor, and hydrogen bond donor, which play important roles in its interaction with the protein targets. Therefore, cryptolepine HCl appears to be a promising drug candidate for further optimization and validation against M. tuberculosis.
    Genetics and molecular research: GMR 09/2015; 14(3):10390-403. DOI:10.4238/2015.September.1.6 · 0.78 Impact Factor
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    • "M. tuberculosis PKnB (EC - is a trans-membrane Ser/Thr protein kinase (STPK) highly conserved in Gram-positive bacteria and apparently essential for mycobacterial viability [3]. Earlier it was shown that PKnB is regulated by auto-phosphorylation an dephosphorylation by the Ser/Thr protein phosphatase [4] and [5] and recent work showed that PKnB is predominantly expressed during exponential growth, where as its over expression causes morphological changes linked to defects in cell wall synthesis and cell division [6]. Aberrant kinase activity is implicated in numerous human diseases and, not surprisingly, protein kinases represent today one of the most important groups of drug targets [7] and [8]. "
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    ABSTRACT: Ser/Thr Kinase is one of the four M. tuberculosis kinases that are conserved in the downsized genome of Mycobacterium leprae and are therefore presumed to play an important role in the processes that regulate the complex life cycle of mycobacteria. It is known that there are two main superfamilies of protein kinases, one including STPKs1 and PTKs and that of His kinases. For a long time, the former were only found in eukaryotes, and the latter were only found in prokaryotes. In this paradigm, proteins from each superfamily were supposed to play analogous roles in the essentially different organization of signal transduction in both phyla. In this study we report the binding mode of Ser/Thr kinase with derivatives of Betulin ((lup-20(29)-ene-3β,28-diol) on the basis of structural similarity, substructure, isomers & conformers. Molecular docking approach using Lamarckian Genetic Algorithm was carried out to find out the potent inhibitors for Ser/Thr Kinase on the basis of calculated ligand-protein pairwise interaction energies. Study was carried out on 3000 molecules which were virtually screened from different databases on the basis of the structural similarity of Betulin. The grid maps representing the protein were calculated using auto grid and grid size was set to 60*60*60 points with grid spacing of 0.375 Ǻ. Docking was carried out with standard docking protocol on the basis of a population size of 150 randomly placed individuals; a maximum number of 2.5 *107 energy evaluations, a mutation rate of 0.02, a crossover rate of 0.80 and an elitism value of 1. Fifteen independent docking runs were carried out for each ligand and results were clustered according to the 1.0 Ǻ rmsd criteria. The docking result of the study of 3000 molecules demonstrated that the binding energies were in the range of -12.72 kcal/mol to -1.71 kcal/mol, with the minimum binding energy of –12.72 kcal/mol. 6 molecules showing hydrogen bonds with the active site residue VAL 95. Further in-vitro and in-vivo study is required on these molecules as the binding mode provided hints for the future design of new derivatives with higher potency and specificity.
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    • "There are few experimentally characterized phosphatases in M. tuberculosis. These include Rv3214 and Rv2419c, which are histidine phosphatases [3,17], PtpA and PtpB which are tyrosine protein phosphatases [41,42], and PstP, a serine/threonine protein phosphatases [43]. The specific substrates of these phosphatases have not been identified yet, with the exception of Rv2419c, a glucosyl-3-phosphoglycerate phosphatase [17]. "
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    ABSTRACT: Functional characterization of genes in important pathogenic bacteria such as Mycobacterium tuberculosis is imperative. Rv2135c, which was originally annotated as conserved hypothetical, has been found to be associated with membrane protein fractions of H37Rv strain. The gene appears to contain histidine phosphatase motif common to both cofactor-dependent phosphoglycerate mutases and acid phosphatases in the histidine phosphatase superfamily. The functions of many of the members of this superfamily are annotated based only on similarity to known proteins using automatic annotation systems, which can be erroneous. In addition, the motif at the N-terminal of Rv2135c is 'RHA' unlike 'RHG' found in most members of histidine phosphatase superfamily. These necessitate the need for its experimental characterization. The crystal structure of Rv0489, another member of the histidine phosphatase superfamily in M. tuberculosis, has been previously reported. However, its biochemical characteristics remain unknown. In this study, Rv2135c and Rv0489 from M. tuberculosis were cloned and expressed in Escherichia coli with 6 histidine residues tagged at the C terminal. Characterization of the purified recombinant proteins revealed that Rv0489 possesses phosphoglycerate mutase activity while Rv2135c does not. However Rv2135c has an acid phosphatase activity with optimal pH of 5.8. Kinetic parameters of Rv2135c and Rv0489 are studied, confirming that Rv0489 is a cofactor dependent phosphoglycerate mutase of M. tuberculosis. Additional characterization showed that Rv2135c exists as a tetramer while Rv0489 as a dimer in solution. Most of the proteins orthologous to Rv2135c in other bacteria are annotated as phosphoglycerate mutases or hypothetical proteins. It is possible that they are actually phosphatases. Experimental characterization of a sufficiently large number of bacterial histidine phosphatases will increase the accuracy of the automatic annotation systems towards a better understanding of this important group of enzymes.
    BMC Microbiology 12/2013; 13(1):292. DOI:10.1186/1471-2180-13-292 · 2.73 Impact Factor
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