Cargando…
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....
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8606822/ https://www.ncbi.nlm.nih.gov/pubmed/34819927 http://dx.doi.org/10.3389/fmicb.2021.772839 |
_version_ | 1784602417654923264 |
---|---|
author | Yang, Shida An, Xiaonan Gu, Guofeng Yan, Zhenxin Jiang, Xukai Xu, Li Xiao, Min |
author_facet | Yang, Shida An, Xiaonan Gu, Guofeng Yan, Zhenxin Jiang, Xukai Xu, Li Xiao, Min |
author_sort | Yang, Shida |
collection | PubMed |
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 2.5mMMg(2+), 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, GlcNH(2)-1-P, and GlcN(3)-1-P) to form nine sugar nucleotides (dTDP-GlcNH(2), dTDP-GlcN(3), UDP-Glc, UDP-GlcNH(2), UDP-GlcN(3), dUDP-Glc, dUDP-GlcNH(2), and dUDP-GlcN(3)). 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. |
format | Online Article Text |
id | pubmed-8606822 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-86068222021-11-23 Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose Yang, Shida An, Xiaonan Gu, Guofeng Yan, Zhenxin Jiang, Xukai Xu, Li Xiao, Min Front Microbiol Microbiology 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 2.5mMMg(2+), 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, GlcNH(2)-1-P, and GlcN(3)-1-P) to form nine sugar nucleotides (dTDP-GlcNH(2), dTDP-GlcN(3), UDP-Glc, UDP-GlcNH(2), UDP-GlcN(3), dUDP-Glc, dUDP-GlcNH(2), and dUDP-GlcN(3)). 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. Frontiers Media S.A. 2021-11-08 /pmc/articles/PMC8606822/ /pubmed/34819927 http://dx.doi.org/10.3389/fmicb.2021.772839 Text en Copyright © 2021 Yang, An, Gu, Yan, Jiang, Xu and Xiao. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Yang, Shida An, Xiaonan Gu, Guofeng Yan, Zhenxin Jiang, Xukai Xu, Li Xiao, Min Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose |
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_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_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_sort | novel dtdp-l-rhamnose synthetic enzymes (rmlabcd) from saccharothrix syringae cgmcc 4.1716 for one-pot four-enzyme synthesis of dtdp-l-rhamnose |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8606822/ https://www.ncbi.nlm.nih.gov/pubmed/34819927 http://dx.doi.org/10.3389/fmicb.2021.772839 |
work_keys_str_mv | AT yangshida noveldtdplrhamnosesyntheticenzymesrmlabcdfromsaccharothrixsyringaecgmcc41716foronepotfourenzymesynthesisofdtdplrhamnose AT anxiaonan noveldtdplrhamnosesyntheticenzymesrmlabcdfromsaccharothrixsyringaecgmcc41716foronepotfourenzymesynthesisofdtdplrhamnose AT guguofeng noveldtdplrhamnosesyntheticenzymesrmlabcdfromsaccharothrixsyringaecgmcc41716foronepotfourenzymesynthesisofdtdplrhamnose AT yanzhenxin noveldtdplrhamnosesyntheticenzymesrmlabcdfromsaccharothrixsyringaecgmcc41716foronepotfourenzymesynthesisofdtdplrhamnose AT jiangxukai noveldtdplrhamnosesyntheticenzymesrmlabcdfromsaccharothrixsyringaecgmcc41716foronepotfourenzymesynthesisofdtdplrhamnose AT xuli noveldtdplrhamnosesyntheticenzymesrmlabcdfromsaccharothrixsyringaecgmcc41716foronepotfourenzymesynthesisofdtdplrhamnose AT xiaomin noveldtdplrhamnosesyntheticenzymesrmlabcdfromsaccharothrixsyringaecgmcc41716foronepotfourenzymesynthesisofdtdplrhamnose |