Jiang Yin-Yin, Tan Yu-Ping, Sun Shu-Fu, Yang Jian, Guo Juan, Tang Jin-Fu
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2022 Aug;47(15):4074-4083. doi: 10.19540/j.cnki.cjcmm.20220509.101.
The lignan glycosyltransferase UGT236(belonging to the UGT71 B family) from Isatis indigotica can catalyze the production of phloridzin from phloretin in vitro. UGT236 shares high identity with P2'GT from apple. In this study, the recombinant plasmid pET28 a-MBP-UGT236 was transferred into Escherichia coli Rosetta(DE3) cells and induced by isopropyl-β-D-thiogalactoside(IPTG). The purified UGT236 protein was used for enzymatic characterization with phloretin as substrate. The results showed that UGT236 had the optimal reaction temperature of 40 ℃ and the optimal pH 8(Na_2HPO_4-NaH_2PO_4 system). The UGT236 activity was inhibited by Ni(2+) and Al(3+), enhanced by Fe(2+), Co(2+), and Mn(2+), and did not affected by Mg(2+), Ca(2+), Li+, Na+, or K+. The K_m, K_(cat), and K_(cat)/K_m of phloretin were 61.03 μmol·L(-1), 0.01 s(-1), and 157.11 mol(-1)·s(-1)·L, and those of UDPG were 183.6 μmol·L(-1), 0.01 s(-1), and 51.91 mol(-1)·s(-1)·L, respectively. The possible active sites were predicted by homologous modeling and molecular docking. By mutagenisis and catalytic activity detection, three key active sites, Glu391, His15, and Thr141, were identified, while Phe146 was related to product diversity. In summary, we found that the lignan glycosyltransferase UGT236 from I.indigotica could catalyze the reaction of phloretin into phloridzin. Several key amino acid residues were identified by structure prediction, molecular docking, and site-mutagenesis, which provided a basis for studying the specificity and diversity of phloretin glycoside products. This study can provide a reference for artificially producing glycosyltransferase elements with high efficiency and specific catalysis.
来自菘蓝的木脂糖基转移酶UGT236（属于UGT71B家族）能够在体外催化根皮素生成根皮苷。UGT236与苹果中的P2'GT具有高度同源性。在本研究中，重组质粒pET28a-MBP-UGT236被转入大肠杆菌Rosetta(DE3)细胞，并经异丙基-β-D-硫代半乳糖苷（IPTG）诱导。以根皮素为底物，对纯化后的UGT236蛋白进行酶学特性分析。结果表明，UGT236的最佳反应温度为40℃，最佳pH值为8（Na₂HPO₄-NaH₂PO₄体系）。UGT236的活性受到Ni²⁺和Al³⁺的抑制，Fe²⁺、Co²⁺和Mn²⁺可增强其活性，而Mg²⁺、Ca²⁺、Li⁺、Na⁺或K⁺对其活性无影响。根皮素的K_m、K_cat和K_cat/K_m分别为61.03μmol·L⁻¹、0.01s⁻¹和157.11mol⁻¹·s⁻¹·L，UDPG的分别为183.6μmol·L⁻¹、0.01s⁻¹和51.91mol⁻¹·s⁻¹·L。通过同源建模和分子对接预测了可能的活性位点。通过诱变和催化活性检测，确定了三个关键活性位点Glu391、His15和Thr141，而Phe146与产物多样性有关。综上所述，我们发现菘蓝中的木脂糖基转移酶UGT236能够催化根皮素转化为根皮苷。通过结构预测、分子对接和位点诱变确定了几个关键氨基酸残基，为研究根皮素糖苷产物的特异性和多样性提供了依据。本研究可为高效、特异性催化的糖基转移酶元件的人工生产提供参考。
