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Functional characterization of the UDP-xylose biosynthesis pathway in Rhodothermus marinus

Applied Microbiology and Biotechnology volume 99, pages 9463–9472 (2015)Cite this article

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. 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.

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Acknowledgments

The authors thank Professor Yuanchao Wang for access to the Bruker Ultraflex MALDI–TOF mass spectrometer and Dr. Ting Wang for her comments on an earlier version of this manuscript. This work was supported by the NJAU Academian- and Departmental Startup Initiative (to both to L.L. and J.V.) and the 100 Foreign Talents Plan (grant number JSB2014012 to J.V.).

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The authors declare that they have no conflict of interest.

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    Affiliations

    1. Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China

      Xu C. Duan, Bin Gu, Zhi P. Cai, Shuang Wei, Pedro Laborda, Li Liu & Josef Voglmeir

    2. College of Sciences, Nanjing Agricultural University, Nanjing, China

      Ai M. Lu

    3. Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China

      Hong Y. Ma

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    1. Xu C. Duan
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    2. Ai M. Lu
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    Correspondence to Li Liu or Josef Voglmeir.

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    Xu C. Duan and Ai M. Lu contributed equally to this work.

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    Duan, X.C., Lu, A.M., Gu, B. et al. Functional characterization of the UDP-xylose biosynthesis pathway in Rhodothermus marinus . Appl Microbiol Biotechnol 99, 9463–9472 (2015). https://doi.org/10.1007/s00253-015-6683-1

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    • DOI: https://doi.org/10.1007/s00253-015-6683-1

    Keywords

    • Rhodothermus marinus
    • UDP-glucuronic acid
    • UDP-xylose
    • UDP-glucose dehydrogenase
    • UDP-xylose synthase