The Role of Arg13 in Protein Phosphatase M tPphA from Thermosynechococcus elongatus

A highly conserved arginine residue is close to the catalytic center of PPM/PP2C-type protein phosphatases. Different crystal structures of PPM/PP2C homologues revealed that the guanidinium side chain of this arginine residue can adopt variable conformations and may bind ligands, suggesting an important role of this residue during catalysis. In this paper, we randomly mutated Arginine 13 of tPphA, a PPM/PP2C-type phosphatase from Thermosynechococcus elongatus, and obtained 18 different amino acid variants. The generated variants were tested towards p-nitrophenyl phosphate and various phosphopeptides. Towards p-nitrophenyl phosphate as substrate, twelve variants showed 3–7 times higher Km values than wild-type tPphA and four variants (R13D, R13F, R13L, and R13W) completely lost activity. Strikingly, these variants were still able to dephosphorylate phosphopeptides, although with strongly reduced activity. The specific inability of some Arg-13 variants to hydrolyze p-nitrophenyl phosphate highlights the importance of additional substrate interactions apart from the substrate phosphate for catalysis. The properties of the R13 variants indicate that this residue assists in substrate binding. 1. Introduction The protein serine/threonine phosphatases constitute two large families, the phosphoprotein phosphatases (PPP) and the metal-dependent protein phosphatases (PPM) and one small family, the aspartate-based phosphatases [1]. The human PPM member PP2Cα [2] has been the defining representative of the PPM family, which is therefore also referred as the PP2C family. PP2C phosphatases are widely present in eukaryotes and prokaryotes where they regulate diverse signaling pathways involved in central cellular processes, such as cell proliferation, stress responses, or metabolic activity [3]. Recently, several crystal structures of bacterial and plant PP2Cs were solved, and they all show that five highly conserved aspartate residues constitute a negative charged pocket that coordinates three / (M1, M2, and M3) ions in the catalytic center [4–9]. All three metal ions were proven by mutational analysis of the coordinating Asp residues to be essential for the activity of PP2Cs [2, 10–12]. Recently, we have reported that the third metal (M3) in the catalytic centre of tPphA (a PP2C member from Thermosynechococcus elongatus) takes part in catalysis, presumably by activating a water molecule to act as proton donor for the leaving group [11]. Recently, further regions in the periphery of the catalytic core of tPphA were identified to play roles in substrate recognition: