title: Divalent metal ion-based catalytic mechanism of the Nudix hydrolase Orf153 (YmfB) from Escherichia coli.
abstract: YmfB from Escherichia coli is the Nudix hydrolase involved in the metabolism of thiamine pyrophosphate, an important compound in primary metabolism and a cofactor of many enzymes. In addition, it hydrolyzes (d)NTPs to (d)NMPs and inorganic orthophosphates in a stepwise manner. The structures of YmfB alone and in complex with three sulfates and two manganese ions determined by X-ray crystallography, when compared with the structures of other Nudix hydrolases such as MutT, Ap4Aase and DR1025, provide insight into the unique hydrolysis mechanism of YmfB. Mass-spectrometric analysis confirmed that water attacks the terminal phosphates of GTP and GDP sequentially. Kinetic analysis of binding-site mutants showed that no individual residue is absolutely required for catalytic activity, suggesting that protein residues do not participate in the deprotonation of the attacking water. Thermodynamic integration calculations show that a hydroxyl ion bound to two divalent metal ions attacks the phosphate directly without the help of a nearby catalytic base.
authors.affiliation: Department of Biological Sciences, Konkuk University, 1 Hwayang dong, Gwangjin-gu, Seoul 143-701, Republic of Korea.
authors.forename: Myoung-Ki
authors.initials: MK
authors.lastname: Hong
authors.affiliation: Requimte/Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal.
authors.forename: António J M
authors.initials: AJ
authors.lastname: Ribeiro
authors.affiliation: Department of Biological Sciences, Konkuk University, 1 Hwayang dong, Gwangjin-gu, Seoul 143-701, Republic of Korea.
authors.forename: Jin-Kwang
authors.initials: JK
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, Konkuk University, 1 Hwayang dong, Gwangjin-gu, Seoul 143-701, Republic of Korea.
authors.forename: Ho-Phuong-Thuy
authors.initials: HP
authors.lastname: Ngo
authors.affiliation: Department of Bioscience and Biotechnology and Bio/Molecular Informatics Center, Konkuk University, 1 Hwayang dong, Gwangjin-gu, Seoul 143-701, Republic of Korea.
authors.forename: Jiyoung
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Bioscience and Biotechnology and Bio/Molecular Informatics Center, Konkuk University, 1 Hwayang dong, Gwangjin-gu, Seoul 143-701, Republic of Korea.
authors.forename: Choong Hwan
authors.initials: CH
authors.lastname: Lee
authors.affiliation: Department of Green Life Science, College of Convergence, Sangmyung University, 7 Hongji-dong, Jongno-gu, Seoul 110-743, Republic of Korea.
authors.forename: Yeh-Jin
authors.initials: YJ
authors.lastname: Ahn
authors.affiliation: Requimte/Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal.
authors.forename: Pedro Alexandrino
authors.initials: PA
authors.lastname: Fernandes
authors.affiliation: School of Pharmaceutical Sciences, Center for Cellular and Structural Biology, Sun Yat-sen University, Guangzhou 510006, People's Republic of China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.affiliation: Requimte/Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal.
authors.forename: Maria Joao
authors.initials: MJ
authors.lastname: Ramos
authors.affiliation: Department of Biological Sciences, Konkuk University, 1 Hwayang dong, Gwangjin-gu, Seoul 143-701, Republic of Korea.
authors.forename: Lin-Woo
authors.initials: LW
authors.lastname: Kang
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000378:metabolism; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics
chemical_list: D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D006153:Guanosine Diphosphate; D008345:Manganese; D006160:Guanosine Triphosphate; D011755:Pyrophosphatases; C513850:YmfB protein, E coli
keywords: Nudix hydrolase; YmfB; metal ion-based catalysis; nucleoside triphosphatase