title: A Single Amino Acid Mutation Converts (R)-5-Diphosphomevalonate Decarboxylase into a Kinase.
abstract: The biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids, via the mevalonate pathway is completed by diphosphomevalonate decarboxylase. This enzyme catalyzes the formation of isopentenyl diphosphate through the ATP-dependent phosphorylation of the 3-hydroxyl group of (R)-5-diphosphomevalonate followed by decarboxylation coupled with the elimination of the 3-phosphate group. In this reaction, a conserved aspartate residue has been proposed to be involved in the phosphorylation step as the general base catalyst that abstracts a proton from the 3-hydroxyl group. In this study, the catalytic mechanism of this rare type of decarboxylase is re-investigated by structural and mutagenic studies on the enzyme from a thermoacidophilic archaeonSulfolobus solfataricusThe crystal structures of the archaeal enzyme in complex with (R)-5-diphosphomevalonate and adenosine 5'-O-(3-thio)triphosphate or with (R)-5-diphosphomevalonate and ADP are newly solved, and theoretical analysis based on the structure suggests the inability of proton abstraction by the conserved aspartate residue, Asp-281. Site-directed mutagenesis on Asp-281 creates mutants that only show diphosphomevalonate 3-kinase activity, demonstrating that the residue is required in the process of phosphate elimination/decarboxylation, rather than in the preceding phosphorylation step. These results enable discussion of the catalytic roles of the aspartate residue and provide clear proof of the involvement of a long predicted intermediate, (R)-3-phospho-5-diphosphomevalonate, in the reaction of the enzyme.
authors.affiliation: From the Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601.
authors.forename: Kento
authors.initials: K
authors.lastname: Motoyama
authors.affiliation: the Graduate School of Engineering, Nagasaki University, Bunkyo-machi, Nagasaki, Nagasaki 852-8521.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Unno
authors.affiliation: From the Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601.
authors.forename: Ai
authors.initials: A
authors.lastname: Hattori
authors.affiliation: the Department of Applied Chemistry, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Takaoka
authors.affiliation: the Department of Applied Chemistry, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654.|the Research Center for Advanced Science and Technology, University of Tokyo, Komaba 4-6-1, Meguro, Tokyo 153-8904, and.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ishikita
authors.affiliation: the Institute of Agriculture, Tokyo University of Agriculture and Technology, Saiwaicho 3-5-8, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawaide
authors.affiliation: From the Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yoshimura
authors.affiliation: From the Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, hhemmi@agr.nagoya-u.ac.jp.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hemmi
mesh_terms: D019943:Amino Acid Substitution; D066241:Carbon-13 Magnetic Resonance Spectroscopy; D002262:Carboxy-Lyases / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D010770:Phosphotransferases / Q000737:chemistry*; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology
chemical_list: D010770:Phosphotransferases; D002262:Carboxy-Lyases; C022503:pyrophosphomevalonate decarboxylase
keywords: archaea; crystal structure; decarboxylase; diphosphomevalonate decarboxylase; enzyme catalysis; enzyme mechanism; isoprenoid; kinase; mevalonate pathway; mutagenesis