Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

Institute of Microbial Technology, Council of Scientific and Industrial Research, Sector 39A, Chandigarh 160 036, India.
Biochemical Journal (Impact Factor: 4.4). 07/2008; 415(1):27-33. DOI: 10.1042/BJ20080234
Source: PubMed

ABSTRACT Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

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    • "It has been suggested that the MurD reaction is the step at which PG synthesis is regulated to maintain the relative thickness of the PG layer in Gram-negative bacteria (Walsh et al., 1999). Furthermore , Mycobacterium tuberculosis MurD is a substrate of PknA kinase, and transphosphorylation of MurD might be involved in the regulation of a switch between PG biosynthesis and cell division in mycobacteria (Thakur and Chakraborti, 2008). "
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    ABSTRACT: With antibiotic resistance mechanisms increasing in diversity and spreading among bacterial pathogens, the development of new classes of antibacterial agents against judiciously chosen targets is a high-priority task. The biochemical pathway for peptidoglycan biosynthesis is one of the best sources of antibacterial targets. Within this pathway are the Mur ligases, described in this review as highly suitable targets for the development of new classes of antibacterial agents. The amide ligases MurC, MurD, MurE and MurF function with the same catalytic mechanism and share conserved amino acid regions and structural features that can conceivably be exploited for the design of inhibitors that simultaneously target more than one enzyme. This would provide multi-target antibacterial weapons with minimized likelihood of target-mediated resistance development.
    Molecular Microbiology 08/2014; 94(2). DOI:10.1111/mmi.12758 · 4.42 Impact Factor
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    • "The role of PknA in regulating cell division in mycobacteria was also confirmed in another study showing modulation of FstZ activity by PknA [66]. Furthermore, also cell wall synthetic enzymes such as MurD, GlmU, and PbpA appear to be regulated by PknA or PknB, supporting the strong relation existing between STPK-dependent phosphorylation and peptidoglycan biosynthesis in cell elongation [67-69]. "
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    ABSTRACT: Recent genetic, biochemical and structural studies have established that eukaryotic-like Ser/Thr protein-kinases are critical mediators of developmental changes and host pathogen interactions in bacteria. Although with lower abundance compared to their homologues from eukaryotes, Ser/Thr protein-kinases are widespread in gram-positive bacteria. These data underline a key role of reversible Ser/Thr phosphorylation in bacterial physiology and virulence. Numerous studies have revealed how phosphorylation/dephosphorylation of Ser/Thr protein-kinases governs cell division and cell wall biosynthesis and that Ser/Thr protein kinases are responsible for distinct phenotypes, dependent on different environmental signals. In this review we discuss the current understandings of Ser/Thr protein-kinases functional processes based on structural data.
    Current Protein and Peptide Science 12/2012; 13(8). DOI:10.2174/138920312804871201 · 3.15 Impact Factor
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    • "In most bacterial proteomes, however, it is difficult to predict which proteins are phosphorylated and which processes are in fact regulated by phosphorylation of Ser/Thr/Tyr. It is also not clear which kinases are involved in the phosphorylation of respective proteins, although a few protein kinases that were found to phosphorylate specific proteins have been identified [16] [17] [18] [19] [20] [21]. Development of phosphoproteomics has enabled us to determine phosphorylation sites of proteins precisely. "
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    ABSTRACT: We performed phosphoproteome analysis of proteins from the extremely thermophilic Gram-negative eubacterium Thermus thermophilus HB8 using gel-free mass spectrometric method. We identified 52 phosphopeptides from 48 proteins and determined 46 phosphorylation sites: 30 on serine, 12 on threonine, and 4 on tyrosine. The identified phosphoproteins are known to be involved in a wide variety of cellular processes. To help elucidate the functional roles of these phosphorylation events, we mapped the phosphorylation sites on the known tertiary structures of the respective proteins. In all, we succeeded in mapping 46 sites (approximately 88%) on the corresponding structures. Most of the phosphorylation sites were found to be located on loops and terminal regions of the secondary structures. Surprisingly, 28 of these sites were situated at or near the active site of the enzyme. In particular, 18 sites were within 4 Å of the ligand, including substrate or cofactor. Such structural locations suggest direct effects of the phosphorylation on the binding of ligand in addition to inducing a conformational change. Interestingly, 19 of these 28 phosphorylation sites were situated near the phosphate moiety of a substrate or cofactor. In oligomeric proteins, 5 phosphorylation sites were found at the subunit interface. Based on these results, we propose a regulatory mechanism that involves Ser/Thr/Tyr phosphorylation in T. thermophilus HB8.
    Proteomics 05/2012; 12(9):1414-30. DOI:10.1002/pmic.201100573 · 3.81 Impact Factor
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