AUTHOR=Yang Shida , An Xiaonan , Gu Guofeng , Yan Zhenxin , Jiang Xukai , Xu Li , Xiao Min TITLE=Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose JOURNAL=Frontiers in Microbiology VOLUME=12 YEAR=2021 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.772839 DOI=10.3389/fmicb.2021.772839 ISSN=1664-302X ABSTRACT=

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 Ss-rmlABCD from Saccharothrix syringae CGMCC 4.1716 and expressed them in Escherichia coli. 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 2.5mMMg²⁺, and the K_m and k_cat values for dTTP and Glc-1-P were 49.56μM and 5.39s⁻¹, and 117.30μM and 3.46s⁻¹, 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, GlcNH₂-1-P, and GlcN₃-1-P) to form nine sugar nucleotides (dTDP-GlcNH₂, dTDP-GlcN₃, UDP-Glc, UDP-GlcNH₂, UDP-GlcN₃, dUDP-Glc, dUDP-GlcNH₂, and dUDP-GlcN₃). Ss-RmlB showed maximal enzyme activity at 50°C and pH 7.5 with 0.02mM NAD⁺, and the K_m and k_cat values for dTDP-glucose were 98.60μM and 11.2s⁻¹, 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.