School of Pharmacy Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China; National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China; School of Pharmaceutical Science, Capital Medical University, Beijing 100069, China.
School of Pharmaceutical Science, Capital Medical University, Beijing 100069, China; School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China.
Chin J Nat Med. 2019 Aug;17(8):575-584. doi: 10.1016/S1875-5364(19)30059-7.
3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.
3-羟-3-甲基戊二酰辅酶 A 合酶（HMGS）是甲羟戊酸途径中的第一个关键酶，参与雷公藤萜类化合物的生物合成。TwHMGS 的全长 cDNA 和 515bp RNAi 靶片段被连接到 pH7WG2D 和 pK7GWIWG2D 载体中，分别进行过表达和沉默，TwHMGS 通过弹道式基因枪转化在雷公藤悬浮细胞中过表达和沉默，与空载体对照相比，表达水平增加了 2 倍，下降到 70%。在 TwHMGS 过表达过程中，TwHMGR、TwDXR 和 TwTPS7v2 的表达明显上调至对照水平。在 RNAi 组中，TwHMGR、TwDXS、TwDXR 和 TwMCT 的表达明显下调至对照水平。过表达 TwHMGS 的细胞产生的三萜内酯产量是对照的两倍。这些结果证明，TwHMGS 的差异表达决定了雷公藤中雷公藤内酯的产生，并在萜类生物合成途径中导致相对基因表达的不同趋势。最后，将乙酰辅酶 A 底物对接到 TwHMGS 的活性位点，表明包括 His247、Lys256 和 Arg296 在内的关键残基与乙酰辅酶 A 发生静电或 H 键相互作用。
