title: Cloning, sequencing, purification, and crystal structure of Grenache (Vitis vinifera) polyphenol oxidase.
abstract: The full-length cDNA sequence (P93622_VITVI) of polyphenol oxidase (PPO) cDNA from grape Vitis vinifera L., cv Grenache, was found to encode a translated protein of 607 amino acids with an expected molecular weight of ca. 67 kDa and a predicted pI of 6.83. The translated amino acid sequence was 99%, identical to that of a white grape berry PPO (1) (5 out of 607 amino acid potential sequence differences). The protein was purified from Grenache grape berries by using traditional methods, and it was crystallized with ammonium acetate by the hanging-drop vapor diffusion method. The crystals were orthorhombic, space group C222(1). The structure was obtained at 2.2 A resolution using synchrotron radiation using the 39 kDa isozyme of sweet potato PPO (PDB code: 1BT1 ) as a phase donor. The basic symmetry of the cell parameters (a, b, and c and alpha, beta, and gamma) as well as in the number of asymmetric units in the unit cell of the crystals of PPO, differed between the two proteins. The structures of the two enzymes are quite similar in overall fold, the location of the helix bundles at the core, and the active site in which three histidines bind each of the two catalytic copper ions, and one of the histidines is engaged in a thioether linkage with a cysteine residue. The possibility that the formation of the Cys-His thioether linkage constitutes the activation step is proposed. No evidence of phosphorylation or glycoslyation was found in the electron density map. The mass of the crystallized protein appears to be only 38.4 kDa, and the processing that occurs in the grape berry that leads to this smaller size is discussed.
authors.affiliation: BG 10 RM 12C206, MSC 1906 National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Virador
authors.forename: Juan P
authors.initials: JP
authors.lastname: Reyes Grajeda
authors.forename: Alejandro
authors.initials: A
authors.lastname: Blanco-Labra
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Mendiola-Olaya
authors.forename: Gary M
authors.initials: GM
authors.lastname: Smith
authors.forename: Abel
authors.initials: A
authors.lastname: Moreno
authors.forename: John R
authors.initials: JR
authors.lastname: Whitaker
mesh_terms: D000595:Amino Acid Sequence; D004156:Catechol Oxidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D027843:Vitis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D010940:Plant Proteins; D004156:Catechol Oxidase