BG 10 RM 12C206, MSC 1906 National Institutes of Health, Bethesda, Maryland 20892, USA.
J Agric Food Chem. 2010 Jan 27;58(2):1189-201. doi: 10.1021/jf902939q.
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.
葡萄多酚氧化酶（PPO）全长 cDNA（P93622_VITVI）序列来自酿酒葡萄（Vitis vinifera L.， cv Grenache），编码一个 607 个氨基酸的翻译蛋白，预计分子量约为 67 kDa，理论等电点为 6.83。翻译的氨基酸序列与白葡萄浆果 PPO（1）（607 个氨基酸潜在序列差异中有 5 个）完全相同。使用传统方法从 Grenache 葡萄浆果中纯化该蛋白，并通过悬滴蒸汽扩散法用醋酸铵结晶。晶体为正交晶系，空间群 C222(1)。使用同步辐射，以甘薯 PPO 的 39 kDa 同工酶（PDB 代码：1BT1）作为相位供体，在 2.2 A 分辨率下获得结构。两种蛋白质的晶体参数（a、b 和 c 以及 alpha、beta 和 gamma）基本对称以及单位晶胞中不对称单位的数量存在差异。两种酶的结构在整体折叠、核心处的螺旋束位置以及活性位点非常相似，在活性位点中，三个组氨酸结合两个催化铜离子中的每一个，其中一个组氨酸与半胱氨酸残基形成硫醚键。提出了 Cys-His 硫醚键的形成构成激活步骤的可能性。在电子密度图中没有发现磷酸化或糖基化的证据。结晶蛋白的分子量似乎仅为 38.4 kDa，并讨论了导致这种较小尺寸的葡萄浆果中的加工过程。
