title: Structure and mechanism of the 2',3' phosphatase component of the bacterial Pnkp-Hen1 RNA repair system.
abstract: Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5' kinase, a central 2',3' phosphatase and a C-terminal ligase. The phosphatase module is a Mn(2+)-dependent phosphodiesterase-monoesterase that dephosphorylates 2',3'-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn(2+) and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn(2+) in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Li Kai
authors.initials: LK
authors.lastname: Wang
authors.forename: Paul
authors.initials: P
authors.lastname: Smith
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D048013:Acetivibrio thermocellus / Q000201:enzymology; D008780:Methyltransferases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D008780:Methyltransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010744:Phosphoric Monoester Hydrolases