School of Life Sciences, Beijing University of Chinese Medicine, Beijing 102488, China; School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
Gene. 2025 Jan 15;933:148919. doi: 10.1016/j.gene.2024.148919. Epub 2024 Sep 3.
Asarum sieboldii Miq. is an important medicinal plant valued for its diverse health benefits in the pharmaceutical industry. In the present study, we isolated and characterized isoeugenol synthase from A. sieboldii (AsIGS), an essential enzyme involved in the biosynthesis of volatile phenylpropenes. We hoped to elucidate the secondary metabolic network of eugenol in A. sieboldii plants, which constructed the prerequisite for quality improvement of the well-known TCM Asari Radix et Rhizoma. Bioinformatics analysis revealed high similarity between the DNA sequences of AsIGS and isoeugenol synthase genes from other plants, and that the association of the candidate protein AsIGS with the PIP reductase family. Moreover, the AsIGS protein displayed a molecular weight of about 34.96 kDa, with a theoretical isoelectric point of 6.01 and an average hydrophobicity of -0.092, indicating the protein's partial acidity, stability, and hydrophilic nature. Phylogenetic analysis showed that AsIGS had a close relationship with isoeugenol synthases and fewer eugenol synthases found in other species. Alphafold2 predicted the structure of the AsIGS protein, and CB-Dock2 predicted the binding sites of the ASIGS-NADPH-coniferyl acetate ternary complex. In vitro enzymatic assay results demonstrated that the optimal temperature of the AsIGS-involved catalysis for coniferyl acetate was 30 °C, and several kinetics parameters were Km (12.21 mM), Vmax (27.9 U/mg), kcat (76.26 s), and kcat/Km (6.49 s·mM). Furthermore, it was also determined that the AsIGS protein had varying performance at different pH levels. While the candidate protein converted coniferyl acetate into both isoeugenol and eugenol at pH 5.5, it just catalyzed the production of isoeugenol at pH 6.5. However, isoeugenol has never been detected in A. sieboldii. Altering AsIGS expression in transgenic plants impacted only eugenol contents. Compared with wild type, overexpression of AsIGS increased eugenol content by 23.3 %, while RNAi-induced down-regulation of AsIGS decreased it by 25.3 %. Taken together, these results confirmed that the AsIGS gene was involved in the biosynthesis of eugenol in A. sieboldii with a dual catalytic potential.
Asarum sieboldii Miq. 是一种重要的药用植物，在制药行业因其多种健康益处而备受重视。在本研究中，我们从 A. sieboldii（AsIGS）中分离和鉴定了异丁香酚合酶，这是一种参与挥发性苯丙烯生物合成的必需酶。我们希望阐明 A. sieboldii 植物中丁香酚的次生代谢网络，为著名的中药细辛根和根茎的质量提高奠定基础。生物信息学分析表明，AsIGS 的 DNA 序列与其他植物的异丁香酚合酶基因高度相似，候选蛋白 AsIGS 与 PIP 还原酶家族有关。此外，AsIGS 蛋白的分子量约为 34.96 kDa，理论等电点为 6.01，平均疏水性为-0.092，表明该蛋白具有部分酸性、稳定性和亲水性。系统发育分析表明，AsIGS 与异丁香酚合酶关系密切，而与其他物种中的丁香酚合酶关系较少。Alphafold2 预测了 AsIGS 蛋白的结构，CB-Dock2 预测了 ASIGS-NADPH-松柏醇乙酸酯三元复合物的结合位点。体外酶促测定结果表明，AsIGS 参与催化松柏醇乙酸酯的最适温度为 30°C，并且几个动力学参数为 Km（12.21 mM）、Vmax（27.9 U/mg）、kcat（76.26 s）和 kcat/Km（6.49 s·mM）。此外，还确定了 AsIGS 蛋白在不同 pH 值下的性能不同。虽然候选蛋白在 pH 5.5 时将松柏醇乙酸酯转化为异丁香酚和丁香酚，但在 pH 6.5 时仅催化异丁香酚的生成。然而，异丁香酚从未在 A. sieboldii 中检测到。改变转基因植物中的 AsIGS 表达仅影响丁香酚的含量。与野生型相比，AsIGS 的过表达使丁香酚含量增加了 23.3%，而 RNAi 诱导的 AsIGS 下调使其降低了 25.3%。综上所述，这些结果证实了 AsIGS 基因参与了 A. sieboldii 中丁香酚的生物合成，具有双重催化潜力。
