Jia Kai-Zhi, Zhu Li-Wen, Qu Xudong, Li Shengying, Shen Yuemao, Qi Qingsheng, Zhang Youming, Li Yue-Zhong, Tang Ya-Jie
Hubei Key Laboratory of Industrial Microbiology, Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Key Laboratory of Fermentation Engineering (Ministry of Education) , Hubei University of Technology , Wuhan 430068 , China.
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences , Wuhan University , Wuhan 430071 , China.
ACS Synth Biol. 2019 Dec 20;8(12):2718-2725. doi: 10.1021/acssynbio.9b00318. Epub 2019 Dec 10.
The 4--β-d-glucopyranoside of DMEP ((-)-4'-desmethylepipodophyllotoxin) (GDMEP), a natural product from , is the direct precursor to the topoisomerase inhibitor etoposide, used in dozens of chemotherapy regimens for various malignancies. The biosynthesis pathway for DMEP has been completed, while the enzyme for biosynthesizing GDMEP is still unclear. Here, we report the enzymatic -glycosylation of DMEP with 53% conversion by exploring the substrate promiscuity and entrances of glycosyltransferases. Notably, we found 6 essential amino acid residues surrounding the putative substrate entrances exposed to the protein surface in UGT78D2, UGT78D2, and UGT78D2-like, and these residues may determine substrate specificity and high -glycosylation activity toward DMEP. Our results provide an effective route for one-step synthesis of GDMEP. Identification of the key residues and entrances of glycosyltransferases will promote precise identification of glycosyltransferase biocatalysts for novel substrates and provide a rational basis for glycosyltransferase engineering.
DMEP（（-）-4'-去甲基表鬼臼毒素）的4-β-D-吡喃葡萄糖苷（GDMEP）是一种天然产物，是拓扑异构酶抑制剂依托泊苷的直接前体，依托泊苷用于多种恶性肿瘤的数十种化疗方案。DMEP的生物合成途径已完成，但生物合成GDMEP的酶仍不清楚。在此，我们通过探索糖基转移酶的底物选择性和入口，报道了DMEP的酶促糖基化反应，转化率为53%。值得注意的是，我们在UGT78D2、UGT78D2和类UGT78D2中发现，围绕假定底物入口的6个必需氨基酸残基暴露于蛋白质表面，这些残基可能决定底物特异性以及对DMEP的高糖基化活性。我们的结果为GDMEP的一步合成提供了一条有效途径。糖基转移酶关键残基和入口的鉴定将促进针对新底物的糖基转移酶生物催化剂的精确鉴定，并为糖基转移酶工程提供合理依据。
