Serine/threonine phosphatases of the PPM 2 family are widely present in eukaryotes and prokaryotes and regulate key signaling pathways involved in cell proliferation, stress responses, or metabolic control (1). PPM phosphatases are metalloenzymes requiring Mg 2ϩ or Mn 2ϩ ions, which are coordinated by a universally conserved core of aspartate residues.The human PPM member PP2C␣ has been the defining representative of this family. As shown by structural analysis and site-directed mutagenesis studies, a binuclear metal center (with metals M1 and M2) activates a catalytic water molecule for nucleophilic attack of the phosphate group (2, 3). The importance of the M1-M2 core was also demonstrated for other PPM members, such as BA-Stp1, a bacterial PPM member from Bacillus anthracis (4). Recently, the structure of several bacterial members (tPphA from Thermosynechococcus elongatus (5), MtPstP from Mycobacterium tuberculosis (6), MspP from Mycobacterium smegmatis (7), STP from Streptococcus agalactiae (8), as well as the structure of Hab1 from Arabidopsis thaliana in co-crystal with its inhibitor, abscisic acid receptor Pyl2 (9)) has been solved. A third metal ion (M3) in proximity of the M1-M2 core could be revealed in several crystal forms of these proteins; however, the function of the third metal was controversially discussed. It was proposed to directly take part in catalysis in the case of SaSTP (8) or to have a regulatory role in the conformation of the flexible FLAP subdomain, which may be important for substrate recognition (10).The PPM tPphA from the thermophilic cyanobacterium T. elongatus is ideally suited to elucidate the role of M3. Because of its thermophilic origin, it has a robust structure, and the M3 coordination was almost invariant in various crystal forms ( Fig. 1) (5), whereas M3 coordination was variable in other PPM members (7,8). Furthermore, tPphA reacts readily with the artificial substrate pNPP, and moreover, its natural substrate, the phosphorylated P II signal transduction protein (P II -P), is available (11). In this study, mutant tPphA variants affected in M3 coordination were generated and analyzed with respect to enzymatic properties, metal binding, and three-dimensional structures, demonstrating a catalytic role of M3 in the dephosphorylation reaction. EXPERIMENTAL PROCEDURESDetailed protocols are available in the supplemental material.Cloning, Overexpression, and Purification of tPphA and Its Variants-The tPphA gene was cloned into the His tag vector pET15b (Novagen) according to standard procedures. Site-directed mutagenesis of tPphA was carried out with the QuickChange XL site-directed mutagenesis kit (Stratagene).Artificial genes for human PP2C fragment (residues 1-297) and the corresponding PP2C-variant D146A were synthesized and cloned into His tag pET15b vector by GENEART (Regensburg, Germany). Overexpression and purification of Histagged proteins were performed as described in the supplemental material.His tag-free tPphA variants D119A and D193A for crystallization were cons...