Koul Anil, Choidas Axel, Tyagi Anil K, Drlica Karl, Singh Yogendra, Ullrich Axel
Department of Molecular Biology, Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany5.
Department of Biochemistry, University of Delhi South Campus, N. Delhi, India3.
Microbiology (Reading). 2001 Aug;147(Pt 8):2307-2314. doi: 10.1099/00221287-147-8-2307.
Pathogenesis of Mycobacterium tuberculosis is closely connected to its survival and replication within the host. Some pathogenic bacteria employ protein kinases that interfere with the cellular signalling network of host cells and promote bacterial survival. In this study, the pknF and pknG genes, which encode two putative protein kinases of M. tuberculosis H(37)Rv, protein kinase F (PknF) and protein kinase G (PknG), respectively, were cloned and expressed in Escherichia coli. Purified PknF phosphorylated the peptide substrate myelin basic protein (MBP) at serine and threonine residues, while purified PknG phosphorylated only at serine residues. The activity of the two kinases was abrogated by mutation of the codon for the predicted ATP-binding-site lysine residue. Southern blot analysis revealed that homologues of the genes encoding the two kinases are present in M. tuberculosis H(37)Ra and Mycobacterium bovis BCG, but not in Mycobacterium smegmatis. Immunoblot analysis of various cellular fractions of M. tuberculosis H(37)Rv revealed that PknF is a transmembrane protein and that PknG is predominantly a cytosolic enzyme. The present study should aid in elucidating the role of these protein kinases in the pathogenesis of mycobacteria.
结核分枝杆菌的发病机制与其在宿主体内的存活和复制密切相关。一些病原菌利用蛋白激酶干扰宿主细胞的细胞信号网络并促进细菌存活。在本研究中，编码结核分枝杆菌H(37)Rv的两种假定蛋白激酶，即蛋白激酶F（PknF）和蛋白激酶G（PknG）的pknF和pknG基因，分别在大肠杆菌中克隆并表达。纯化的PknF在丝氨酸和苏氨酸残基处使肽底物髓鞘碱性蛋白（MBP）磷酸化，而纯化的PknG仅在丝氨酸残基处磷酸化。预测的ATP结合位点赖氨酸残基的密码子突变消除了这两种激酶的活性。Southern印迹分析表明，编码这两种激酶的基因同源物存在于结核分枝杆菌H(37)Ra和牛分枝杆菌卡介苗中，但不存在于耻垢分枝杆菌中。对结核分枝杆菌H(37)Rv各种细胞组分的免疫印迹分析表明，PknF是一种跨膜蛋白，而PknG主要是一种胞质酶。本研究应有助于阐明这些蛋白激酶在分枝杆菌发病机制中的作用。
