黄素单加氧酶 VibMO1 的酶学性质及其分子改造

	黄素单加氧酶 VibMO1 的酶学性质及其分子改造
	戚芳婷
导师	曾英
关键词	黄素单加氧酶，VibMO1，酶学性质，催化机理，分子改造 Flavin monooxygenase, VibMO1, Enzymatic properties, Catalytic mechanism, Molecular engineering
摘要	对苯二酚类混源萜是一类以对苯二酚及其衍生物作为非萜结构单元的混源萜，这类天然产物广泛存在于自然界中例如辅酶 Q，也有物种特异的次生代谢产物比如紫草素。对苯二酚类混源萜具有多种多样的化学结构与生物活性，其生物合成通常需要进行对羟基苯甲酸结构单元的脱羧羟化，但在真核生物中相关酶的催化机理未见报道。 VibMO1 是首个从高等真菌褐盖韧革菌（Boreostereum vibrans）中鉴定到的催化氧化脱羧关键反应具有普适性的黄素单加氧酶（flavin monooxygenase），该酶催化异戊烯基对羟基苯甲酸发生脱羧羟化反应生成异戊烯基对苯二酚。与原核生物辅酶 Q 生物合成中对羟基苯甲酸结构单元的氧化脱羧需两种酶分步完成不同，VibMO1 可同时催化脱羧与羟化，并且发生在芳环的同一碳原子上。该酶的天然底物异戊烯基对羟基苯甲酸与紫草素前体香叶基对羟基苯甲酸的结构非常相似，但紫草素的生物合成途径中参与脱羧羟化步骤的关键酶还未见报道。因此，开展 VibMO1 酶学特性与催化机理的研究，不仅可以加深对黄素单氧酶的认识，还将有助于阐明紫草素和真核细胞辅酶 Q 生物合成中相关反应的酶分子机制。为了揭示 VibMO1 催化脱羧羟化反应的机理，扩大黄素单加氧酶在生物医药领域的应用，本论文综合运用生物信息学与天然产物分离纯化、异源表达与酶活检测、同源建模与分子对接、氨基酸定点突变等技术对 VibMO1 酶学性质、催化机理进行研究，并对其进行分子改造。主要结果如下： 1、VibMO1 酶学性质的测定。VibMO1 在氧化脱羧过程中利用 NAD(P)H 作为电子供体，对 NADH 具有偏好性。VibMO1 脱羧羟化的最适温度为 28 ℃，最适反应缓冲液为 pH=7.5 的 25 mM Hepes 缓冲液，动力学常数 Km 值为 0.419 ? 0.086 mM，kcat 为 14.935 ? 0.050 min-1。底物特异性实验结果表明 VibMO1 仅对在苯核 C4 位具有羟基取代基的苯甲酸衍生物才有活性，并且 VibMO1 可使紫草素生物合成前体香叶基对羟基苯甲酸脱羧羟化。 2、VibMO1 催化机理研究。VibMO1 的蛋白结构与 A 类黄素单加氧酶整体结构非常相似，脱羧羟化反应中的氧原子来源于氧气。本文根据分子对接结果与定点突变实验确定了反应过程中的 5 个关键氨基酸：Tyr228、His224、Pro314、Arg114 与 Arg192。研究结果表明，无底物时 Arg114 与 FAD 长链上 C2、C4 上氧有疏水作用即与 FAD 形成氢键以稳定 FAD。当底物到达活性位点时，His224和Pro314 与底物羧基形成氢键，形成并稳定 VibMO1-底物复合体，Tyr228 使底物去质子化促进芳环电子重排，同时底物诱导 FAD 发生构象变化，Arg192 与FAD 形成氢键使 FAD 稳定于“out”构象，FAD 被 NADH 还原后回到“in”构象，活性位点构象的变化使 FAD 的 C4a 接近底物苯环的 C1 原子，允许 Fl4a-OOH攻击底物 C1 使其发生羟基化，随之脱去羧基生成对苯二酚结构单元。 3、VibMO1 的分子改造。为提高 VibMO1 对香叶基对羟基苯甲酸的脱羧活性，本文以扩大活性口袋为手段进行分子改造，获得了高性能突变酶 H217A，H217A 对香叶基对羟基苯甲酸的脱羧活性较野生型 VibMO1 提高了 16 倍。; Meroterpenoids containing (hydro)quinone derivatives as non-terpenoid moieties are widely distributed or species-specific natural products，such as coenzyme Q and shikonin. They have diverse structures and a wide range of biological activities. The biosynthetic origin of (hydro)quinone moieties are known to occur via decarboxylation and hydroxylation of 4-hydroxybenzoate unit, however, no catalytic mechanism of the corresponding enzyme has been reported in eukaryotes. VibMO1 is the first flavin monooxygenase that converts 3-prenyl-4-hydroxybenzoate into prenylhydroquinone in the biosynthesis of (hydro)quinonecontaining meroterpenoids in Boreostereum vibrans. Unlike the oxidative decarboxylation in prokaryotic coenzyme Q pathway that often requires two enzymes to remove the carboxyl group and insert the hydroxyl group, VibMO1 can perform simultaneous decarboxylation and hydroxylation of 3-prenyl-4-hydroxybenzoate at the same aromatic carbon atom to generate prenylhydroquinone. Furthermore, the shikonin biosynthetic pathway also involves oxidative decarboxylation of a 4-hydroxybenzoate unit, but the corresponding enzyme remains unknown. Therefore, studies on the enzymatic properties and catalytic mechanism of VibMO1 can not only expand the understanding of flavin monooxygenases and their application in the biomedical field, but also provide some insight into the molecular mechanism involved in the biosynthesis of shikonin and coenzyme Q in eukaryotes. Here, we elucidated the enzymatic properties, catalytic mechanism and altered the molecular structure of VibMO1 using a combination of bioinformatics, the extraction and purification of natural products, heterogeneous expression, molecular docking and site-directed mutagenesis. The major results are listed below： 1. Enzymatic properties. VibMO1, a FAD-binding monooxygenase, need NAD(P)H and notably prefer NADH as the electron donor in catalysis reaction. Km is 0.419 ? 0.086 mM, and kcat is 14.935 ? 0.050 min-1 in pH 7.5 Hepes buffer (25 mM) at 28 ℃. In substrate specificity experiments, VibMO1 can only catalyze benzoic acid derivatives with hydroxyl group at the C4 site of benzene ring. It is worth nothing that VibMO1 is able to catalyze 3-geranyl hydroxybenzoic acid (GBA) to obtain geranylhydroquinone, the precursor of shikonin. 2. Catalytic mechanism. The mass-spectral analysis of prenylhydroquinone showed that substrate is oxidized by ambient O2. Molecular docking studies with predicted protein structure were performed to design 15 mutants of VibMO1. Interestingly, 5 mutants including R114A, R192A, H224A, Y228A and P314A are entirely inactive, proving that these five amino acid sites play a key role in the catalytic reaction. The docking results indicate that His224 and Pro314 may stabilize the substrate in a suitable position for rapid reaction with flavin. The deprotonation of substrate relies on the Tyr228 for providing hydrogen bond to the hydroxy group of the substrate.
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75107
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	戚芳婷. 黄素单加氧酶 VibMO1 的酶学性质及其分子改造[D]. 中国科学院大学,2022.

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