STABILIZATION OF THE PHOSPHO-ASPARTYL RESIDUE IN A 2-COMPONENT SIGNAL-TRANSDUCTION SYSTEM IN THERMOTOGA-MARITIMA

The central signaling pathway in many bacterial regulatory systems inv olves phosphotransfer between two conserved proteins, a histidine prot ein kinase, and a response regulator. The occurrence of two-component signaling systems in thermophilic bacteria raises questions of how bot h 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 res ponse regulator. Both DrrA and the cytoplasmic region of HpkA (HpkA57) have been expressed in Escherichia coli, purified, and characterized. HpkA57 and DrrA have apparent T-m's of 75 and 90 degrees C, respectiv ely. HpkA57 exhibits ATP-dependent autophosphorylation activity simila r to that of histidine kinases from mesophiles, with maximum activity at 70 degrees C. DrrA catalyzes transfer of phosphoryl groups from Hpk A57 and exhibits Mg2+-dependent autophosphatase activity, with maximum activity at approximately 80 degrees C. 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 degrees C. In the absence of Mg2+, at a ll temperatures examined, phospho-DrrA exhibits much greater stability than acetyl phosphate. This suggests that the active site of this hyp erthermophilic response regulator is designed to protect the phospho-a spartyl residue from hydrolysis.