鉴定丹参中的clerodane二萜修饰细胞色素P450(CYP728D26)--精神药物丹参素A的生物合成途径。

IF 5.4 2区 生物学 Q1 PLANT SCIENCES Physiologia plantarum Pub Date : 2024-09-01 DOI:10.1111/ppl.14569
Iris Ngo, Rahul Kumar, Liang Li, Seon-Won Kim, Moonhyuk Kwon, Dae-Kyun Ro
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引用次数: 0

摘要

丹参是一种原产于墨西哥瓦哈卡的致幻植物。这种植物产生精神作用的活性成分是丹参素 A,它是一种强效、高选择性的κ-阿片受体激动剂。丹参素 A 与吗啡和可待因等其他众所周知的阿片类药物不同,它是一种非氮二萜类化合物,对生物碱类阿片的主要受体μ-阿片受体没有亲和力。萜类阿片选择性地靶向一种新的阿片受体(κ-阿片受体),有助于开发替代镇痛药。阐明丹皮酚 A 的生物合成途径有助于生物制造丹皮酚 A 的各种半合成衍生物,但迄今为止,丹皮酚 A 途径中只有两种酶被鉴定出来。在这里,我们确定了 CYP728D26,它能催化巴豆内酯 G 的 C18 氧合反应,巴豆内酯 G 具有一个烯丙基二烷骨架。通过酵母体内重组、纯化产物的 1H 和 13C-NMR 分析以及 CYP728D26 的动力学分析(Km 值为 13.9 μM),验证了 CYP728D26 的生化特性。除了 C18 上的一次加氧反应外,碰撞诱导解离分析表明,CYP728D26 还催化了另外两次加氧反应,形成巴豆苷 G 酸,尽管这种羧酸形式是次要产物。其近亲 CYP728D25 在重组酵母系统中表现出对缬草烷骨架的 C1-羟基化。然而,CYP728D25 在体外试验中没有表现出活性。这一结果表明,应谨慎解释从超表达系统中观察到的催化活性。这项工作鉴定了一种新的 CYP 催化剂,并增进了我们对丹参素 A 生物合成的了解。
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Identification of clerodane diterpene modifying cytochrome P450 (CYP728D26) in Salvia divinorum - en route to psychotropic salvinorin A biosynthesis.

Salvia divinorum is a hallucinogenic plant native to the Oaxaca in Mexico. The active ingredient for psychotropic effects in this plant is salvinorin A, a potent and highly selective κ-opioid receptor agonist. Salvinorin A is distinct from other well-known opioids, such as morphine and codeine, in that it is a non-nitrogenous diterpenoid with no affinity for μ-opioid receptor, the prime receptor of alkaloidal opioids. A terpene opioid that selectively targets a new opioid receptor (κ-opioid receptor) can be instrumental in developing alternative analgesics. Elucidation of the salvinorin A biosynthetic pathway can help bio-manufacture diverse semi-synthetic derivatives of salvinorin A but, to date, only two enzymes in the Salvinorin A pathway have been identified. Here, we identify CYP728D26 that catalyzes a C18 oxygenation on crotonolide G, which bears a clerodane backbone. Biochemical identity of CYP728D26 was validated by in vivo reconstitution in yeast, 1H- and 13C-NMR analyses of the purified product, and kinetic analysis of CYP728D26 with a Km value of 13.9 μM. Beyond the single oxygenation on C18, collision-induced dissociation analysis suggested two additional oxygenations are catalyzed by CYP728D26 to form crotonoldie G acid, although this carboxylic acid form is a minor product. Its close homologue CYP728D25 exhibited a C1-hydroxylation on the clerodane backbone in a reconstituted yeast system. However, CYP728D25 showed no activity in in vitro assays. This result implies that catalytic activities observed from overexpression systems should be interpreted cautiously. This work identified a new CYP catalyst and advanced our knowledge of salvinorin A biosynthesis.

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来源期刊
Physiologia plantarum
Physiologia plantarum 生物-植物科学
CiteScore
11.00
自引率
3.10%
发文量
224
审稿时长
3.9 months
期刊介绍: Physiologia Plantarum is an international journal committed to publishing the best full-length original research papers that advance our understanding of primary mechanisms of plant development, growth and productivity as well as plant interactions with the biotic and abiotic environment. All organisational levels of experimental plant biology – from molecular and cell biology, biochemistry and biophysics to ecophysiology and global change biology – fall within the scope of the journal. The content is distributed between 5 main subject areas supervised by Subject Editors specialised in the respective domain: (1) biochemistry and metabolism, (2) ecophysiology, stress and adaptation, (3) uptake, transport and assimilation, (4) development, growth and differentiation, (5) photobiology and photosynthesis.
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