三叶崖爬藤肉桂酸-4-羟化酶的生物信息学及体外功能研究

Functional characterization of cinnamate 4-hydroxylase from Tetrastigma hemsleyanum: an in silico and in vitro study

肉桂酸-4-羟化酶(C4H)是苯丙烷代谢途径的关键酶之一,在植物黄酮类物质的合成代谢过程中发挥着重要的作用。在受到低温胁迫时中药材三叶崖爬藤(Tetrastigma hemsleyanum)中具有药用价值的黄酮类物质总含量上升,但其机制尚不清楚。本研究通过实时荧光定量PCR (qRT-PCR)分析低温胁迫下三叶崖爬藤C4H (ThC4H)基因表达量上升的现象,并利用c DNA末端快速扩增(RACE)技术获得ThC4H基因的cDNA全长,随后在酿酒酵母中重组表达,检测其体外活性,并进行三维结构比对和底物分子对接研究。结果表明:该cDNA具有完整的开放阅读框1 518 bp,编码505个氨基酸。ThC4H具有C4H家族保守的P450结构域,与花叶地锦(Parthenocissus henryana)的C4H有91.88%的相似性。重组ThC4H蛋白具有体外催化反式肉桂酸生成4-香豆酸的能力。ThC4H、苔藓植物C4H和高粱(Sorghum bicolor) C4H (SbC4H1)与肉桂酸对接位置及形成的氢键基本一致,猜测其催化机制相似,而导致不同C4H酶性质差异的可能原因是2号环(loop)区域和血红素结合域中2个氨基酸的多样性。本研究为进一步探索三叶崖爬藤ThC4H蛋白在抗寒方面的功能提供参考。

Cinnamate 4-hydroxylase(C4H), a key enzyme in the phenylpropanoid metabolism of plants,plays an important role in the flavonoid biosynthetic pathway. Cold stress could significantly increase the total content of flavonoids which exhibit various pharmacological activities in Tetrastigma hemsleyanum.However, the exact mechanism is still unclear. In this study, the results of real-time quantitative PCR(qRTPCR) showed that the relative expression of C4H in T. hemsleyanum(ThC4H) increased under cold stress.Therefore, the full-length cDNA sequence of ThC4H was obtained by rapid-amplification of cDNA ends(RACE) and cloned into Saccharomyces cerevisiae. The activity of recombinant protein ThC4H was analyzed in vitro. The structural alignment and the molecular docking with substrate were also studied. The results showed that the cDNA of ThC4H contained an open reading frame of 1 518 bp, encoding 505 amino acids. Moreover, the ThC4H contained a conserved P450 domain and shared 91.88% sequence identity with the C4H of Parthenocissus henryana. Recombinant ThC4H displayed the ability to catalyze trans-cinnamic acid to p-coumaric acid in vitro. Molecular docking results showed that the formation of hydrogen bonds and the binding site location and orientation of C4H to trans-cinnamic acid were similar among T.hemsleyanum, liverwort and Sorghum bicolor, suggesting that they probably share a similar catalytic mechanism. Furthermore, the loop of number 2 and the diversity of two residues in the heme binding domain might contribute to the different properties of C4H. These results laid a foundation for the further functional exploration of C4H under low-temperature stress in T. hemsleyanum.