Table1_Functional characterization of CYP96T1-like cytochrome P450 from Lycoris aurea catalyzing para-para′ and para-ortho′ oxidative coupling in Amaryllidaceae alkaloids biosynthesis.xlsx

Amaryllidaceae alkaloids (AAs) are complex plant secondary metabolites possessing a wide range of biological activities. 4′-O-methylnorbelladine (4OMN) is the branchpoint intermediate for the entire AAs, and was the last common intermediate before AA pathway branches diverge. The cyclization of 4OMN by C-C oxidative coupling, which can afford para-para′, ortho-para′, and para-ortho′ scaffold, was catalyzed by cytochrome P450 96T (CYP96T) family enzymes. To clarify the mechanisms involved in this controversial step, four CYP96T homologs (LauCYP96T1, LauCYP96T1-like-1, LauCYP96T1-like-2 and LauCYP96T1-like-3) were cloned from the full-length transcriptome of Lycoris aurea. All the four LauCYP96T are localized to endoplasmic reticulum. Functional analysis reveals that LauCYP96T1 and LauCYP96T1-like proteins display inverted regioselectivity for oxidative coupling of 4OMN, in which LauCYP96T1 and LauCYP96T1-like-2 dominantly afford para-para′ scaffold, and LauCYP96T1-like-1 and LauCYP96T1-like-3 are responsible for para-ortho′ scaffold formation. Using molecular homology modeling and docking studies, we predicted models for the binding of 4OMN to LauCYP96T, and identified two amino acid residues that might be responsible for the dominant changes in generated products of para-ortho′ and para-para′ oxidative coupling. Our results highlight the functional diversity and promiscuity of LauCYP96T enzymes and might provide valuable information for Amaryllidaceae alkaloid production.

石蒜科生物碱（Amaryllidaceae alkaloids, AAs）是一类结构复杂的植物次生代谢产物，具有多样的生物活性。4′-O-甲基去甲雪波罗定（4′-O-methylnorbelladine, 4OMN）是整条石蒜科生物碱生物合成通路的分支节点中间体，亦是该通路各分支分化前的最后一个共同中间体。由细胞色素P450 96T（cytochrome P450 96T, CYP96T）家族酶催化的4OMN碳-碳氧化偶联环化反应，可生成对-对′、邻-对′及对-邻′三种核心骨架结构。为阐明这一存在争议的反应步骤的分子机制，研究团队从忽地笑（Lycoris aurea）的全长转录组中克隆获得4个CYP96T同源基因，分别为LauCYP96T1、LauCYP96T1-like-1、LauCYP96T1-like-2与LauCYP96T1-like-3。亚细胞定位分析显示，这4种LauCYP96T酶均定位于内质网。功能验证结果表明，LauCYP96T1与LauCYP96T1-like蛋白对4OMN的氧化偶联反应呈现反转的区域选择性：其中LauCYP96T1和LauCYP96T1-like-2主要催化生成对-对′骨架，而LauCYP96T1-like-1与LauCYP96T1-like-3则负责介导对-邻′骨架的形成。本研究通过分子同源建模与分子对接实验，预测了4OMN与LauCYP96T的结合模型，并鉴定出两个可能决定产物偏好性转变的氨基酸残基，该转变对应对-邻′与对-对′氧化偶联产物的生成偏好切换。本研究结果凸显了LauCYP96T家族酶的功能多样性与催化混杂特性，可为石蒜科生物碱的生物合成生产提供有价值的理论参考。