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Mechanism of signal transduction in a model representative of prokaryotic heme-containing oxygen sensors
Smrčka, Tomáš ; Martínková, Markéta (advisor) ; Stráňava, Martin (referee)
A two-component system, consisting of a histidine kinase and a response regulator, is a crucial molecular-biology tool for many bacteria to react to an environmental changes. An important step in activation of the two-component system is an autophosphorylation reaction on the dimeric histidine kinase, which involves the transfer of a phosphate group from ATP in the catalytic domain to a conserved histidine molecule. Depending on whether the transfer of the phosphate group occurs within one subunit of the dimer or from one subunit to another, we distinguish a cis- or trans-autophosphorylation, respectively. Here we study the autophosphorylation reaction of globin coupled histidine kinase from soil bacteria Anaeromyxobacter sp. Fw109-5 (AfGcHK), which uses heme to detect gaseous molecules. Using a phosphorylation analysis of a heterodimer of AfGcHK composed of a subunit with a defective ATP-binding site and a subunit with a phosphorylatable histidine substituted for alanine, the trans-mechanism of autophosphorylation was identified for AfGcHK. Key words: two component signal systems, histidine kinase, heme-containing oxygen sensors, Phos-tag, AfGcHK [IN CZECH]
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The kinetic analysis of the enzyme reaction catalyzed by the globine coupled histidine kinase
Fojtíková, Veronika ; Martínková, Markéta (advisor) ; Vaněk, Ondřej (referee)
Two-component signal systems serve as basic stimulus-response coupling mechanism to allow organisms (predominantly bacteria) to sense and respond to changes in many environmental conditions. The prototypical system consists of two proteins, namely a histidine kinase, containing a sensor domain and catalytic kinase core, and a response regulator protein (RR protein). Extracellular stimuli are sensed by a histidine kinase sensor domain. Then ATP is bound to the catalytic kinase core and the γ-phosphoryl group is transferred to the conserved histidine residue. This phosphoryl group is subsequently transferred to a conserved aspartate residue within the RR protein. Phosphotransfer to the RR protein results in activation of a downstream effector domain that elicits the specific response (usually it is transcription activity, but a few RR proteins function as enzymes). The histidine kinase sensor domain is designed for specific ligand interactions. This master thesis focused on the unique histidine kinase containing a sensor domain with a globine structure, which coordinates a heme molecule, namely globin-coupled histidine kinase from Anaeromyxobacter sp. Fw 109-5 (AfGcHK) and its appropriate RR protein. The aim of this thesis was to study and characterize the phosphorylation activity of AfGcHK and RR...
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