| Summary: | Tuberculosis is a major cause of premature mortality world wide. Nevertheless, we have very limited understanding of the physiology of its causative agent M. tuberculosis. This thesis focuses on M. tuberculosis PknH kinase and signal transduction cascade mediated by it. PknH kinase is a fascinating kinase to study because it was shown to mediate reduced M. tuberculosis growth in vivo through sensing of nitric oxide. Identification of PknH signal transduction cascade can lead to better understanding of M. tuberculosis physiology during infection. The only known phosphorylation substrate of PknH to date is a transcription factor EmbR. It is know to regulate transcription of cell wall production genes. It seemed unlikely that EmbR, the only known PknH substrate, alone can mediate such complex reaction during infection. Therefore, we hypothesized that PknH kinase must phosphorylates multiple substrates, other than EmbR. And the phosphorylation sites on the substrates are similar to the autophosphorylation site on PknH kinase. We were able to visualize 9 PknH specific phospho-proteins by two-dimensional phosphor-proteome analysis. Alternatively, using a bioinformatic approach we predicted 40 potential PknH phosphorylation substrates. Two of the predicted substrates were selected for further studies. In vitro kinase studies concluded both Rv0681 and DacB1 are phosphorylated by PknH kinase, and point mutation studies confirmed that PknH kinase phosphorylates both substrates at predicted sites. We also performed enzyme kinetic studies to compare PknH phosphorylation of 3 different substrates. The results revealed that PknH is able to phosphorylate Rv0681 and DacB1 with much higher enzyme velocity than EmbR; but EmbR exhibited much higher Km value than Rv0681 and DacB1. The results presented in this thesis suggest that DacB1 and Rv0681 are true phosphorylation targets of PknH kinase; and the interaction between kinase and substrates are more complex than we initially thought. === Medicine, Faculty of === Medicine, Department of === Experimental Medicine, Division of === Graduate
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