Discovery And Functional Characterization Of The UDP-xylose Biosynthesis Pathway Enzymes Originated From Rhodothermus Marinus

Nucleotide sugar is the active monosugar donor for glycosyltransfering reaction to form the glycochain.Xylose(Xyl)is one of the key monosaccharide building blocks for many glycoconjugates and the terminal Xyl has been found to play various essential roles in the livings.For examples,Xyl participates in synthesizing glycosaminoglycans which are widely involved in cell proliferation,cell migration,cell adhesion,cell communication,cancer growth and pathogen invasion in animals.In plants and microoganisms,xyl is essential part for many cell wall structures or lipid polysaccharides and so on.UDP-Xylose is the nucleotide donor of Xyl for the synthesis of Xyl-containing substances.It is therefore of great importance to synthesize UDP-Xyl for the addition of Xyl to different substrates.Enzymatic synthesis for nucleotide sugar is a more preferable method than chemical method,due to the mild reaction condition,high purity of product and less pollution.The pathway for UDP-Xyl biosynthesis has been revealed,and UGD and UXS are the two enzymes responsible for the production of UDP-Xyl starting from low cost UDP-Glc through the middle product of UDP-GlcA.Our study was focused on this pathway and aimed to discover novel UGD and UXS enzymes from thermomyces bacteria for UDP-Xyl synthesis.In this study,we cloned,expressed and characterized two UGD genes,RmUGDl and RmUGD2,and one UXS gene,RmUXS from Rhodothermus marinus which is living in hot spring water.The results showed RmUGD1 and RmUXS were highly active and thermostable which confer them great potential for being applied in industrial production of UDP-Glc A and UDP-Xyl.Details of the results are showed below:1.Cloning and sequence analysis of the genes RmUGDl,RmUGD2 and RmUXS Two UGD genes and one UXS gene named RmUGDl,RmUGD2 and RmUXS were cloned from Rhodothermus marinus.Sequence analysis showed that these three genes are highly conserved among diverse organisms,with some share even higher than 50%of similarity.However,the similarity between RmUGDl and RmUGD2 is only 29%.Protein domains prediction found that RmUGD1 and RmUGD2 both have an N-terminal and a C-terminal SDR structure,but RmUGD lacks another UDPG-MGDP-dehydrogenase domain in N-terminal region.RmUXS has an extended SDR structure which contains an NAD+ binding domain and two substrate binding domains.Besides,SigIP and TMHMM analysis did not reveal any signal peptides or transmembrane structures from these three proteins.2.Protein expression and activity test of RmUGDl,RmUGD2 and RmUXSAfter expression and purification,SDS-PAGE assay showed clear bands for RmUGD1,RmUGD2 and RmUXS,with each having the same molecular weight as estimated in theory.UPLC analysis of the reaction products demonstrated that RmUGD1 and RmUXS were both active but RmUGD2 was not.Heating-purification of RmUXS exhibited the same quality as His-Tag purification,which indicated a simpler and efficient purification method.Both Real-Time NMR and MALDI-TOF-MS were employed to confirm the RmUXS reaction through detecting the production of UDP-Xyl.The UDP-Xyl proton signal was gradually increasing while UDP-GlcA signal was decreasing during the reaction.Both results showed the production of UDP-Xyl increased with time and the substrate UDP-GlcA was completely used and converted within 10-15 min.3.Biochemical characterization of recombinant RmUGDlThe optimum pH of RmUGDl is 9.0 and the optimum temperature was determined as 60℃.Metal ions are not essential for enzymatic activity,however,Zn2+ and Co2+ ions decreased the activity of RmUGD1 and Mg2+ increased the activity.Other metal ions such as Ca2+,Cu2+,Fe2+,Fe2+,Mn2+ and Ni2+ had no significant effect on recombinant RmUGD1.Addition of 100 mM and 250 mM NaCl slightly enhanced the activity of RmUGD1,but higher than 250 mM of NaCl started to inhibit the enzyme.Addition of SDS(2 mM)nearly completely inhibited the activity of RmUGD1,while the addition of Triton-X100(2 mM)slightly enhanced the activity of the enzyme.The enzyme was stable at 70℃for at least 3 hours but easy to lose the activity at 80℃within 1 hour.The activity of the enzyme was clearly inhibited by UDP-xylose as a negative feedback effect.RmUGD1 Km value for NAD+ was 25μM,Vmax value was 3.3μM min-1 and Kcat value was 0.032 min-1.For UDP-glucuronic acid,the Km value was determined at 33μM,Vmax was 3.4μM min-1 and the Kcat value was 0.030 min-1.4.Biochemical characterization of recombinant RmUXSThe optimal pH of RmUXS was between pH 7.5 and the optimal temperature was 60℃.The activity of RmUXS required no metal ions but was inhibited by Zn2+,Ni2+,Co2+and Mn2,and was enhanced by Ca2+,Fe2+ and Fe3.High salt concentrations posed higher effects on the activity of RmUXS than that of RmUGD1.UDP and UTP significantly decreased and SDS completely inhibited the activity of RmUXS.RmUXS was stable at 70℃for at least 3 hours,but was almost completely inactivated after being heated at 80℃for one hour.Km value of RmUXS for UDP-glucuronic acid was 32μM,Vmax value was 10.2μM min-1 and Kcat value was 0.102 min-1.5.UDP-Xyl synthesis in one-pot reactionOne-pot reaction with both RmUGD and RmUXS successfully converted UDP-Glc directly into UDP-Xyl.The best ratio between RmUGDl and RmUXS in the reaction was in a range from 1:0.25 to 1:0.5;the optimal pH of the reaction was 9.0 and the optimal temperature was 60°C.Orthogonal test indicated the best combination for these factors were pH9.0,60℃and 1:1 between RmUGD1 and RmUXS.