Biochemical characterization of aspartyl phosphate phosphatase interaction with a phosphorylated response regulator and its inhibition by a pentapeptide

Abstract

The RapA and RapB proteins are aspartyl phosphate phosphatases that specifically dephosphorylate the Spo0F∼P intermediate response regulator of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The ∼48-kDa His-tag derivative proteins were purified by metal affinity chromatography, and their molecular and biochemical characteristics were studied. RapA and RapB were found to be dimers in solution. Enzymatic activity was strongly dependent upon maintaining reducing conditions during purification and storage. RapA phosphatase activity on Spo0F∼-P is inhibited in vivo by a pentapeptide generated from the phrA gene. Native gel assays demonstrated that the RapA dimer forms a stable complex with two molecules of Spo0F∼P or with its PhrA pentapeptide inhibitor. The pentapeptide was shown to displace Spo0F∼P from a preformed complex with RapA. The structural organization of Rap phosphatases in tetratricopeptide repeats provides insights on the mechanisms of RapA interaction with its substrate and its inhibitor.