title: Functional characterization of the UDP-xylose biosynthesis pathway in Rhodothermus marinus.
abstract: 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 1 h exposure at 70 °C. No metal ions were required for enzymatic activities. Zn(2+) 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.
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Xu C
authors.initials: XC
authors.lastname: Duan
authors.affiliation: College of Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Ai M
authors.initials: AM
authors.lastname: Lu
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Bin
authors.initials: B
authors.lastname: Gu
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Zhi P
authors.initials: ZP
authors.lastname: Cai
authors.affiliation: Department of Plant Pathology, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Hong Y
authors.initials: HY
authors.lastname: Ma
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Wei
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Pedro
authors.initials: P
authors.lastname: Laborda
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People's Republic of China. lichen.liu@njau.edu.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.affiliation: Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People's Republic of China. josef.voglmeir@njau.edu.cn.
authors.forename: Josef
authors.initials: J
authors.lastname: Voglmeir
mesh_terms: D055162:Biocatalysis; D053898:Biosynthetic Pathways; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D045482:Hot Springs / Q000382:microbiology; D007700:Kinetics; D011994:Recombinant Proteins / Q000378:metabolism; D045245:Rhodothermus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014540:Uridine Diphosphate Xylose / Q000096:biosynthesis*; D014994:Xylose / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014535:Uridine Diphosphate Glucuronic Acid; D014540:Uridine Diphosphate Xylose; D014994:Xylose; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase; D014532:Uridine Diphosphate Glucose
keywords: Rhodothermus marinus; UDP-glucose dehydrogenase; UDP-glucuronic acid; UDP-xylose; UDP-xylose synthase