Stabilization of the phospho-aspartyl residue in a two-component signal transduction system in Thermotoga maritima.
The central signaling pathway in many bacterial regulatory systems involves phosphotransfer between two conserved proteins, a histidine protein kinase, and a response regulator. The occurrence of two-component signaling systems in thermophilic bacteria raises questions of how both the proteins and the labile acyl phosphate of the response regulator are adapted to function at elevated temperatures. Thermotoga maritima HpkA is a transmembrane histidine kinase, and DrrA is its cognate response regulator. Both DrrA and the cytoplasmic region of HpkA (HpkA57) have been expressed in Escherichia coli, purified, and characterized. HpkA57 and DrrA have apparent Tm's of 75 and 90 degreesC, respectively. HpkA57 exhibits ATP-dependent autophosphorylation activity similar to that of histidine kinases from mesophiles, with maximum activity at 70 degreesC. DrrA catalyzes transfer of phosphoryl groups from HpkA57 and exhibits Mg2+-dependent autophosphatase activity, with maximum activity at approximately 80 degreesC. At this temperature, the half-life for phospho-DrrA is approximately 3 min. In the absence of Mg2+, the half-life is 26 min, significantly greater than the half-life of a typical acyl phosphate at 80 degreesC. In the absence of Mg2+, at all temperatures examined, phospho-DrrA exhibits much greater stability than acetyl phosphate. This suggests that the active site of this hyperthermophilic response regulator is designed to protect the phospho-aspartyl residue from hydrolysis.
Center for Advanced Biotechnology and Medicine, Howard Hughes Medical Institute, Rutgers University, Piscataway, New Jersey 08854, USA.
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Lee
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D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D000071677:Histidine Kinase; D006358:Hot Temperature; D010755:Organophosphates / Q000378:metabolism; D010766:Phosphorylation; D011489:Protein Denaturation; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D013816:Thermodynamics; D020124:Thermotoga maritima / Q000378:metabolism / Q000502:physiology*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C101895:DrrA protein, Bacteria; D010755:Organophosphates; C061278:PhoB protein, Bacteria; C061279:PhoR protein, Bacteria; D001224:Aspartic Acid; C011632:acetyl phosphate; D011494:Protein Kinases; C091973:autophosphorylation-dependent multifunctional protein kinase; D000071677:Histidine Kinase