Enzymatic Synthesis of Trideuterated Sialosides.

Sialic acids are a family of acidic monosaccharides often found on the termini of cell surface proteins or lipid glycoconjugates of higher animals. Herein we describe the enzymatic synthesis of the two isotopically labeled sialic acid derivatives d3-X-Gal-α-2,3-Neu5Ac and d3-X-Gal-α-2,3-Neu5Gc. Using deuterium oxide as the reaction solvent, deuterium atoms could be successfully introduced during the enzymatic epimerization and aldol addition reactions when the sialosides were generated. NMR and mass spectrometric analyses confirmed that the resulting sialosides were indeed tri-deuterated. These compounds may be of interest as internal standards in liquid chromatography/mass spectrometric assays for biochemical or clinical studies of sialic acids. This was further exemplified by the use of this tri-deuterated sialosides as internal standards for the quantification of sialic acids in meat and egg samples.

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

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.