Yang Shida, An Xiaonan, Gu Guofeng, Yan Zhenxin, Jiang Xukai, Xu Li, Xiao Min
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.
National Glycoengineering Research Center, Shandong Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Qingdao, China.
Front Microbiol. 2021 Nov 8;12:772839. doi: 10.3389/fmicb.2021.772839. eCollection 2021.
Deoxythymidine diphospho-l-rhamnose (dTDP-l-rhamnose) is used by prokaryotic rhamnosyltransferases as the glycosyl donor for the synthesis of rhamnose-containing polysaccharides and compounds that have potential in pharmaceutical development, so its efficient synthesis has attracted much attention. In this study, we successfully cloned four putative dTDP-l-rhamnose synthesis genes from CGMCC 4.1716 and expressed them in . The recombinant enzymes, Ss-RmlA (glucose-1-phosphate thymidylyltransferase), Ss-RmlB (dTDP-d-glucose 4,6-dehydratase), Ss-RmlC (dTDP-4-keto-6-deoxy-glucose 3,5-epimerase), and Ss-RmlD (dTDP-4-keto-rhamnose reductase), were confirmed to catalyze the sequential formation of dTDP-l-rhamnose from deoxythymidine triphosphate (dTTP) and glucose-1-phosphate (Glc-1-P). Ss-RmlA showed maximal enzyme activity at 37°C and pH 9.0 with 2.5mMMg, and the and values for dTTP and Glc-1-P were 49.56μM and 5.39s, and 117.30μM and 3.46s, respectively. Ss-RmlA was promiscuous in the substrate choice and it could use three nucleoside triphosphates (dTTP, dUTP, and UTP) and three sugar-1-Ps (Glc-1-P, GlcNH-1-P, and GlcN-1-P) to form nine sugar nucleotides (dTDP-GlcNH, dTDP-GlcN, UDP-Glc, UDP-GlcNH, UDP-GlcN, dUDP-Glc, dUDP-GlcNH, and dUDP-GlcN). Ss-RmlB showed maximal enzyme activity at 50°C and pH 7.5 with 0.02mM NAD, and the and values for dTDP-glucose were 98.60μM and 11.2s, respectively. A one-pot four-enzyme reaction system was developed by simultaneously mixing all of the substrates, reagents, and four enzymes Ss-RmlABCD in one pot for the synthesis of dTDP-l-rhamnose and dUDP-l-rhamnose with the maximal yield of 65% and 46%, respectively, under the optimal conditions. dUDP-l-rhamnose was a novel nucleotide-activated rhamnose reported for the first time.
脱氧胸苷二磷酸 -L- 鼠李糖（dTDP-L- 鼠李糖）被原核鼠李糖基转移酶用作糖基供体，用于合成含鼠李糖的多糖和具有药物开发潜力的化合物，因此其高效合成备受关注。在本研究中，我们成功从中国普通微生物菌种保藏管理中心 4.1716 中克隆了四个假定的 dTDP-L- 鼠李糖合成基因，并在[具体表达宿主未给出]中进行了表达。重组酶 Ss-RmlA（葡萄糖 -1- 磷酸胸苷酰转移酶）、Ss-RmlB（dTDP-D- 葡萄糖 4,6- 脱水酶）、Ss-RmlC（dTDP-4- 酮 -6- 脱氧葡萄糖 3,5- 表异构酶）和 Ss-RmlD（dTDP-4- 酮 - 鼠李糖还原酶）被证实可催化由三磷酸脱氧胸苷（dTTP）和葡萄糖 -1- 磷酸（Glc-1-P）依次形成 dTDP-L- 鼠李糖。Ss-RmlA 在 37°C 和 pH 9.0 以及 2.5 mM Mg 条件下表现出最大酶活性，dTTP 和 Glc-1-P 的 Km 和 Vmax 值分别为 49.56 μM 和 5.39 s，以及 117.30 μM 和 3.46 s。Ss-RmlA 在底物选择上具有宽泛性，它可以使用三种核苷三磷酸（dTTP、dUTP 和 UTP）和三种糖 -1-P（Glc-1-P、GlcNH-1-P 和 GlcN-1-P）形成九种糖核苷酸（dTDP-GlcNH、dTDP-GlcN、UDP-Glc、UDP-GlcNH、UDP-GlcN、dUDP-Glc、dUDP-GlcNH 和 dUDP-GlcN）。Ss-RmlB 在 50°C 和 pH 7.5 以及 0.02 mM NAD 条件下表现出最大酶活性，dTDP- 葡萄糖的 Km 和 Vmax 值分别为 98.60 μM 和 11.2 s。通过将所有底物、试剂和四种酶 Ss-RmlABCD 同时混合在一个反应体系中，开发了一种一锅四酶反应系统用于合成 dTDP-L- 鼠李糖和 dUDP-L- 鼠李糖，在最佳条件下，最大产率分别为 65% 和 46%。dUDP-L- 鼠李糖是首次报道的新型核苷酸活化鼠李糖。
