title: 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 approximately P intermediate response regulator of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The approximately 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 approximately 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 approximately P or with its PhrA pentapeptide inhibitor. The pentapeptide was shown to displace Spo0F approximately 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.
authors.affiliation: Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Shu
authors.initials: S
authors.lastname: Ishikawa
authors.forename: Leighton
authors.initials: L
authors.lastname: Core
authors.forename: Marta
authors.initials: M
authors.lastname: Perego
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D011994:Recombinant Proteins; C445331:aspartyl phosphate phosphatase; D010744:Phosphoric Monoester Hydrolases