caa a22 4500 605500819 CHVBK 20210128100557.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6683-1 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6683-1 Functional characterization of the UDP-xylose biosynthesis pathway in Rhodothermus marinus [Elektronische Daten] [Xu Duan, Ai Lu, Bin Gu, Zhi Cai, Hong Ma, Shuang Wei, Pedro Laborda, Li Liu, Josef Voglmeir] UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS) are the two enzymes responsible for the biosynthesis of UDP-xylose from UDP-glucose. Several UGDs from bacterial sources, which oxidize UDP-glucose to glucuronic acid, have been found and functionally characterized whereas only few reports on bacterial UXS isoforms exist. Rhodothermus marinus, a halothermophilic bacterium commonly found in hot springs, proved to be a valuable source of carbohydrate active enzymes of biotechnological interest, such as xylanases, mannanases, and epimerases. However, no enzymes of R. marinus involved in the biosynthesis or modification of nucleotide sugars have been reported yet. Herein, we describe the cloning and characterization of two putative UGD (RmUGD1 and RmUGD2) and one UXS (RmUXS) isoform from this organism. All three enzymes could be expressed in recombinant form and purified to near homogeneity. UPLC- and NMR-based activity tests showed that RmUGD1 and RmUXS are indeed active enzymes, whereas no enzymatic activity could be detected by RmUGD2. Both RmUGD1 and RmUXS showed a temperature optimum of 60°C, with almost no loss of activity after 1h exposure at 70°C. No metal ions were required for enzymatic activities. Zn2+ ions strongly inhibited both enzymes. RmUGD1 showed higher salt tolerance and had a higher pH optimum than RmUXS. Furthermore, RmUGD1 was inhibited by UDP-xylose at higher concentrations. By coupling recombinant RmUXS and RmUGD1, UDP-xylose could be successfully synthesized directly from UDP-glucose. The high activity of the herein described enzymes make RmUGD1 and RmUXS the first thermo-tolerant biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose. Springer-Verlag Berlin Heidelberg, 2015 Rhodothermus marinus nationallicence UDP-glucuronic acid nationallicence UDP-xylose nationallicence UDP-glucose dehydrogenase nationallicence UDP-xylose synthase nationallicence Duan Xu Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut Lu Ai College of Sciences, Nanjing Agricultural University, Nanjing, China aut Gu Bin Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut Cai Zhi Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut Ma Hong Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China aut Wei Shuang Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut Laborda Pedro Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut Liu Li Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut Voglmeir Josef Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/22(2015-11-01), 9463-9472 0175-7598 99:22<9463 2015 99 253 https://doi.org/10.1007/s00253-015-6683-1 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6683-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Duan Xu Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut NATIONALLICENCE 700 1- Lu Ai College of Sciences, Nanjing Agricultural University, Nanjing, China aut NATIONALLICENCE 700 1- Gu Bin Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut NATIONALLICENCE 700 1- Cai Zhi Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut NATIONALLICENCE 700 1- Ma Hong Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China aut NATIONALLICENCE 700 1- Wei Shuang Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut NATIONALLICENCE 700 1- Laborda Pedro Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut NATIONALLICENCE 700 1- Liu Li Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut NATIONALLICENCE 700 1- Voglmeir Josef Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/22(2015-11-01), 9463-9472 0175-7598 99:22<9463 2015 99 253