黄酮8-O-甲基转移酶功能解析与催化机理研究

Methyltransferases play vital roles in the metabolic pathways of plants and animals. The biological activity of flavonoids could be greatly improved after the active hydroxyl being methylated. Among the plant flavonoid O-methyltransferases that has been reported, most of their substrates are 4′-hydroxyl flavonoids, and the catalytic sites are concentrated in the 3′-, 3′/5′-, 4′- or 7- of the substrates. The flavonoid O-methyltransferase which specifically catalyzes the methylation of 8-hydroxyl in 4′-deoxyflavanone has not been reported yet. In this study, through transcriptome analysis of Scutellaria baicalensis and Scutellaria barbata, flavonoid 8-O-methyltransferase (F8OMT), a key enzyme for the biosynthesis of anti-tumor drug wogonin, will be explored combined with biosynthetic technology. The catalytic mechanism of flavonoid substrate binding domain and the conformational change mechanism of F8OMT will be further explored through homologous modeling, molecular docking and kinetic simulation, and higher catalytic activity mutant will be obtained through semi-rational design. Then, the biosynthesis pathway of wogonin will be reassembled in Saccharomyces cerevisiae and realizes the de novo biosynthesis of wogonin in microbe for the first time. Through this study, the regioselectivity and functional evolution mechanism of flavonoid O-methyltransferase will be elucidated more comprehensively, and also provides important significance for the synthetic biology research of methylated natural products.

甲基转移酶在动植物代谢途径中都起着非常关键的催化作用。黄酮类化合物的活泼羟基经过甲基化修饰后，其生物活性有很大程度的提高。在已发现的植物黄酮O-甲基转移酶中，大多数催化底物是4′位羟基化的黄酮类化合物，且催化位置集中在其3′、3′/5′、4′以及7位。特异性催化4′-脱氧黄酮的8位羟基进行甲基化反应的黄酮O-甲基转移酶尚未见报道。本项目将通过对黄芩和半枝莲进行转录组分析，结合合成生物学技术挖掘抗肿瘤药物汉黄芩素合成关键酶黄酮8-O-甲基转移酶（F8OMT）；通过同源建模、分子对接和动力学模拟深入探究其黄酮底物结合域的催化机理和酶构象改变机制，并通过半理性设计获得高催化活性的突变体；在酿酒酵母体内重构汉黄芩素的生物合成途径，将首次实现汉黄芩素的微生物从头合成。本研究将有助于更全面地阐明黄酮-O-甲基转移酶的区域选择性反应机制和功能进化机制，也对甲基化天然产物的合成生物学研究具有重要意义。

甲基转移酶在动植物代谢过程中起着非常关键的催化作用。黄酮类化合物的活泼羟基经过甲基化修饰后，可以很大程度提高其生物活性和药用价值。汉黄芩素是一种8位甲基化的4′-脱氧黄酮类化合物，其抗肿瘤活性显著。本项目通过对黄芩和半枝莲进行转录组分析，结合合成生物学技术分析挖掘到汉黄芩素合成的关键酶黄酮8-O-甲基转移酶（F8OMT）；通过在酿酒酵母体内重构汉黄芩素的生物合成途径，首次实现汉黄芩素的微生物从头合成；通过对黄酮-O-甲基转移酶进行同源建模、分子对接和结构解析完成其催化机理研究；此外，通过多物种筛选和半理性设计获得高活性的黄酮-O-甲基转移酶突变体，实现多种甲基化黄酮的微生物异源合成。本研究将有助于更全面地阐明黄酮-O-甲基转移酶的区域选择性反应机制和功能进化机制，也对甲基化天然产物的合成生物学研究具有重要意义。