Phenylpropanoid 2,3-dioxygenase involved in the cleavage of the ferulic acid side chain to form vanillin and glyoxylic acid in Vanilla planifolia

Abstract

Enzyme catalyzing the cleavage of the phenylpropanoid side chain was partially purified by ion exchange and gel filtration column chromatography after (NH 4) 2 SO 4 precipitation. Enzyme activities were dependent on the concentration of dithiothreitol (DTT) or glutathione (GSH) and activated by addition of 0.5 mM Fe 2+. Enzyme activity for ferulic acid was as high as for 4-coumaric acid in the presence of GSH, suggesting that GSH acts as an endogenous reductant in vanillin biosynthesis. Analyses of the enzymatic reaction products with quantitative NMR (qNMR) indicated that an amount of glyoxylic acid (GA) proportional to vanillin was released from ferulic acid by the enzymatic reaction. These results suggest that phenylpropanoid 2,3-dioxygenase is involved in the cleavage of the ferulic acid side chain to form vanillin and GA in Vanilla planifolia.

Novel Eco-friendly, One-Pot Method for the Synthesis of Kynurenic Acid Ethyl Esters

“…The method relies on the principle of rationalizing peaks: one (or more) of the analyte to one (or more) of the reference compound (internal standard). Beneficial traits of the adequate internal standard are the easily recognizable peaks in the 1 H NMR spectra, chemical stability in the reaction media, and easy handling. − These requirements led to the selection of p -methoxybenzoic acid (anisic acid, PAA) purchased from Merck as the internal standard. However, due to the complex matrices and, in some cases, low conversions and/or overlapping peaks, the method was found to be not universal (Figure ).…”

Research progress on the biosynthesis of plant phenylpropanoids

“…Route I)。 2009 年 Van Moerkercke 等从矮牵牛(Petunia hybrida)中鉴 定了一个定位于过氧化物酶体的 3-酮脂酰辅酶 A 硫 解酶--PhKAT，并通过基因沉默抑制 PhKAT 表达确 定 了 其 在 苯 甲 醛 生 物 合 成 中 的 作 用 [16] 。 2012 年 Colquhoun 等从矮牵牛中克隆一个酰基辅酶 A 连接酶 (acyl-activating enzyme，AAE)PhAAE [17] 。他们推 测 PhAAE 可能负责在矮牵牛过氧化物酶体中催化肉 桂酸生成肉桂酰辅酶 A，但 没有体外实验证明该酶具 有肉桂酸连接酶活性。 同年另一项研究从矮牵牛中发 现了一 个 定 位 于 过 氧 化 物 酶 体 的 辅酶 A 连接酶 --PhCNL，并通过体外实验确定其可以催化肉桂酸生 成肉桂酸辅酶 A [18] 。2022 年矮牵牛中催化肉桂酰辅 酶 A 水解脱氢的双功能酶 PhCHD 被成功解析出来 [19] 。PhCHD 的催化需要 NAD + 的协助，同时镁离子 可以提高其对肉桂酰辅酶 A 的催化效率。2022 年黄 惺祺等在 矮 牵 牛 中 鉴 定 了 一 个 苯 甲 醛 还 原 酶 PhBS [20] 。不同于其他酰基辅酶 A 还原酶，PhBS 是一 个由 PhBSα 和 PhBSβ 组成的异源二聚体。单独的 PhBSα 和 PhBSβ 都不具有催化活性，只有形成二聚 体才具有催化活性。PhBS 高度特异性识别苯甲酰辅 酶 A，对其他酰基辅酶 A 的催化活性很差。 除了 β 氧化途径， 植物还可能通过其他非 β 氧化 途径生成苯甲醛。 如在细胞质中直接氧化裂解肉桂酸 生成苯甲醛和乙醛酸(图 1. Route II) [21] ，或者先将 肉桂酸水解生成 3-羟基苯丙酸， 再裂解生成苯甲醛和 乙酸(图 1. Route III) [22] 。在过氧化物酶体中可能也 存在直接催化肉桂酰辅酶 A 氧化裂解生成苯甲醛的 途径(图 1.…”

“…A c c e p t e d https://engine.scichina.com/doi/10.1360/SSV-2024-0029 图 1. 植物中苯甲醛生物合成途径 [16][17][18][19][20][21][22] 。实心箭头表示催化反应和对应基因已经确认，虚线箭头表示催化反应已确认但未确 认对应的基因。PAL：苯丙氨酸解氨酶；CHD：肉桂酰辅酶 A 水合/脱氢酶；KAT：3-酮脂酰辅酶 A 硫解酶；BS：苯甲醛合 成酶；3O3PP-CoA：3-氧代-3-苯基丙酰辅酶 A。…”

Is a spice missing from the recipe? The intra‐cellular localization of vanillin biosynthesis needs further investigations

“…In light of the conflicting results, the debate around Vp VAN activity remains. In addition, a second enzyme, named phenylpropanoid 2,3‐dioxygenase (PPDiox), was reported to catalyse cleavage of the ferulic acid side chain to form vanillin in V. planifolia (Negishi & Negishi 2017 ). Remarkably, it appears that proteins from two different protein families can catalyse the same reaction.…”