Plant Biochemistry Laboratory and Centre for Structural-Functional Genomics, Concordia University, Montreal, QC, H4B 1R6, Canada.
BMC Plant Biol. 2010 Jul 29;10:156. doi: 10.1186/1471-2229-10-156.
Wheat (Triticum aestivum L.) O-methyltransferase (TaOMT2) catalyzes the sequential methylation of the flavone, tricetin, to its 3'-methyl- (selgin), 3',5'-dimethyl- (tricin) and 3',4',5'-trimethyl ether derivatives. Tricin, a potential multifunctional nutraceutical, is the major enzyme reaction product. These successive methylations raised the question as to whether they take place in one, or different active sites. We constructed a 3-D model of this protein using the crystal structure of the highly homologous Medicago sativa caffeic acid/5-hydroxyferulic acid O-methyltransferase (MsCOMT) as a template with the aim of proposing a mechanism for multiple methyl transfer reactions in wheat.
This model revealed unique structural features of TaOMT2 which permit the stepwise methylation of tricetin. Substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions. Mutational analysis of structurally guided active site residues identified those involved in binding and catalysis. The partly buried tricetin active site, as well as proximity and orientation effects ensured sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups. We also demonstrate that Val309, which is conserved in a number of graminaceous flavone OMTs, defines the preference of TaOMT2 for tricetin as the substrate.
We propose a mechanism for the sequential methylation of tricetin, and discuss the potential application of TaOMT2 to increase the production of tricin as a nutraceutical. The single amino acid residue in TaOMT2, Val309, determines its preference for tricetin as the substrate, and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT.
小麦（Triticum aestivum L.）O-甲基转移酶（TaOMT2）催化黄酮、tricetin 的顺序甲基化，生成 3'-甲基（selgin）、3'，5'-二甲基（tricetin）和 3'，4'，5'-三甲醚衍生物。tricetin 是一种潜在的多功能营养保健品，是主要的酶反应产物。这些连续的甲基化引发了一个问题，即它们是否发生在一个或不同的活性位点上。我们使用高度同源的 Medicago sativa 咖啡酸/5-羟基阿魏酸 O-甲基转移酶（MsCOMT）的晶体结构作为模板，构建了该蛋白的 3-D 模型，旨在提出小麦中多个甲基转移反应的机制。
该模型揭示了 TaOMT2 的独特结构特征，允许 tricetin 的逐步甲基化。底物结合由广泛的氢键和范德华相互作用介导。结构引导的活性位点残基的突变分析确定了参与结合和催化的残基。部分埋藏的 tricetin 活性位点，以及接近和取向效应，确保了在同一口袋内底物的顺序甲基化。tricetin 的逐步甲基化涉及到其羟基由 His262-Asp263 对脱质子化，然后是 SAM-甲基的亲核攻击。我们还证明，在许多禾本科黄酮 OMT 中保守的 Val309 定义了 TaOMT2 对 tricetin 作为底物的偏好。
我们提出了 tricetin 顺序甲基化的机制，并讨论了 TaOMT2 增加 tricin 作为营养保健品产量的潜在应用。TaOMT2 中的单个氨基酸残基 Val309 决定了它对 tricetin 作为底物的偏好，并且可能定义了两个密切相关的蛋白质，COMT 和黄酮 OMT 之间的进化差异。
