Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose
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....
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Frontiers Media S.A.
2021
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oai:doaj.org-article:472407afd80c4e18b2aee3f0b10829eb2021-11-08T07:53:58ZNovel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose1664-302X10.3389/fmicb.2021.772839https://doaj.org/article/472407afd80c4e18b2aee3f0b10829eb2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmicb.2021.772839/fullhttps://doaj.org/toc/1664-302XDeoxythymidine 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 Km and kcat 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 Km and kcat 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.Shida YangXiaonan AnGuofeng GuGuofeng GuZhenxin YanXukai JiangXukai JiangLi XuLi XuMin XiaoMin XiaoMin XiaoFrontiers Media S.A.articleRmlABCD enzymesSaccharothrix syringaedTDP-l-rhamnosedUDP-l-rhamnoseenzymatic synthesisMicrobiologyQR1-502ENFrontiers in Microbiology, Vol 12 (2021) |
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| collection |
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| topic |
RmlABCD enzymes Saccharothrix syringae dTDP-l-rhamnose dUDP-l-rhamnose enzymatic synthesis Microbiology QR1-502 |
| spellingShingle |
RmlABCD enzymes Saccharothrix syringae dTDP-l-rhamnose dUDP-l-rhamnose enzymatic synthesis Microbiology QR1-502 Shida Yang Xiaonan An Guofeng Gu Guofeng Gu Zhenxin Yan Xukai Jiang Xukai Jiang Li Xu Li Xu Min Xiao Min Xiao Min Xiao Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose |
| description |
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 Km and kcat 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 Km and kcat 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. |
| format |
article |
| author |
Shida Yang Xiaonan An Guofeng Gu Guofeng Gu Zhenxin Yan Xukai Jiang Xukai Jiang Li Xu Li Xu Min Xiao Min Xiao Min Xiao |
| author_facet |
Shida Yang Xiaonan An Guofeng Gu Guofeng Gu Zhenxin Yan Xukai Jiang Xukai Jiang Li Xu Li Xu Min Xiao Min Xiao Min Xiao |
| author_sort |
Shida Yang |
| title |
Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose |
| title_short |
Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose |
| title_full |
Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose |
| title_fullStr |
Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose |
| title_full_unstemmed |
Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose |
| title_sort |
novel dtdp-l-rhamnose synthetic enzymes (rmlabcd) from saccharothrix syringae cgmcc 4.1716 for one-pot four-enzyme synthesis of dtdp-l-rhamnose |
| publisher |
Frontiers Media S.A. |
| publishDate |
2021 |
| url |
https://doaj.org/article/472407afd80c4e18b2aee3f0b10829eb |
| work_keys_str_mv |
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