Cloning, overexpression, and characterization of a serine/threonine protein kinase pknI from Mycobacterium tuberculosis H37Rv.
Protein phosphorylation-dephosphorylation is the principal mechanism for translation of external signals into cellular responses. Eukaryotic-like serine/threonine kinases have been reported to play important roles in bacterial development and/or virulence. The PknI protein is one of the 11 eukaryotic-like serine/threonine kinases in Mycobacterium tuberculosis H37Rv. From the bioinformatic studies, PknI protein has been shown to have an N-terminal cytoplasmic domain followed by a transmembrane region and an extracellular C-terminus suggestive of a sensor molecule. In this study, we have cloned, overexpressed, and characterized the entire coding region and the cytoplasmic domain of PknI as a fusion protein with an N-terminal histidine tag, and used immobilized metal affinity chromatography for purification of recombinant proteins. The purified recombinant proteins were found to be functionally active through in vitro phosphorylation assay and phosphoamino acid analysis. In vitro kinase assay of both proteins revealed that PknI is capable of autophosphorylation and showed manganese-dependent activity. Phosphoamino acid analysis indicated phosphorylation at serine and threonine residues. Southern blot analysis with genomic DNA highlighted the conserved nature of pknI among the various mycobacterial species. In silico analysis revealed a close homology of PknI to Stk1 from Streptococcus agalactiae, shown to have a role in virulence and cell segregation of the organism.
Gopalaswamy
Radha
R
Department of Immunology, Tuberculosis Research Centre (ICMR), Mayor V.R. Ramanathan Road, Chetput, Chennai 600031, India.
Narayanan
P R
PR
Narayanan
Sujatha
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D019295:Computational Biology; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D019606:Phosphoamino Acids / Q000032:analysis; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D019606:Phosphoamino Acids; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases