Stabilization of the Phospho-aspartyl Residue in a Two-Component Signal Transduction System in Thermotoga maritima

Abstract

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.

Kinetic Characterization of the WalRKSpn(VicRK) Two-Component System ofStreptococcus pneumoniae: Dependence of WalKSpn(VicK) Phosphatase Activity on Its PAS Domain

“…This prediction was confirmed by phosphatase assays showing that the half-life of WalR Spn ϳP was Ϸ23 h at 25°C (Table 3, line 1). The WalR Spn ϳP autophosphatase activity was not affected by addition of ADP, similar to other response regulators, including VanR, PhoQ, and DrrA (29,71,94).…”

Matching Biochemical Reaction Kinetics to the Timescales of Life: Structural Determinants That Influence the Autodephosphorylation Rate of Response Regulator Proteins

“…Despite the strongly conserved active sites geometries for the activated states of response regulators, measured rates of autodephosphorylation span almost six orders of magnitude21–23, with faster autodephosphorylation rates correlating with faster biological responses (such as chemotaxis) and slower autodephosphorylation rates correlating with slower biological processes (such as development). How do response regulators, with highly conserved active site geometries and catalytic mechanisms, tailor the kinetics of signaling biochemistry (e.g.…”

Synthesis of [32P]Phosphoramidate for Use as a Low Molecular Weight Phosphodonor Reagent

“…For DrrA, a protein from the moderate hyperthermophile T. maritima, convenient rates of phosphorylation require elevated temperatures (22), and for this protein we carried out phosphorylation at 60°C for 20 min (Fig. 3, Lane 1).…”

“…Bovine pancreatic ribonuclease A (RNase A) was from Amersham Pharmacia Biotech. Thermotoga maritima DrrA protein (22), Salmonella typhimurium CheB and CheB(D56N) (23), and Salmonella typhimurium CheY and CheY(D57N) (24) were produced and purified as previously described. Prior to protein phosphorylation reactions, protein samples were freshly desalted into 50 mM NaH 2 PO 4 /100 mM Na 2 SO 4 pH 7.5 using gelpermeation chromatography, concentrated using Microcon-10 concentrator units, and filtered through 0.2-m cellulose acetate spin filters.…”