Optimization of the Enzymatic Synthesis of UDP‐Xylose and Characterization of Enzymes Involved

Abstract

The plant cell wall is of interest for agriculture and biofuel production; understanding the biosynthesis of plant cell walls is critical for improving the viability of biorenewable energy as a replacement for traditional energy sources. Xyloglucan is an important cell wall constituent and has high degrees of xylosylation. However, studies of xylosylation of hemicelluloses and other biomolecules are hampered, partially due to the low supply of the primary substrate for xylosylation, UDP‐xylose (UDPX). The goal of this project has been to optimize the known biological pathway for UDPX synthesis for laboratory use. The two‐enzyme UDPX biosynthesis pathway produces UDPX from UDP‐glucose (UDPG), a more readily available substrate chemical. The project also characterized enzymatic properties of the enzymes UDP‐glucose dehydrogenase (Ugd) and UDP‐xylose synthase (Uxs), which perform the catalytic activities in the pathway of interest. These enzyme homologs have not been characterized previously.Ugd and Uxs were expressed and purified using the plasmids prepared in Dr. Adam Barb's lab. While homologs of Ugd and Uxs have been partially characterized before, neither has been studied to the degree required for this project and the specific homologs used herein have never been reported. Enzymatic assays were used to determine the optimum conditions for each enzyme and spectrophotometry and high‐performance liquid chromatography were used to measure the products of reactions. Biologically relevant conditions including pH, salt content, reducing agent content, presence of metal ions, et cetera were optimized. Subsequently, methods of combining the reaction systems have been attempted. Due to the significantly different pH optimums of the two enzymes and the generation of inhibitory compounds, the synthesis of UDPX cannot be performed as a one‐pot reaction and will require two independent, consecutive reactions. Additionally, kinetics data collected has provided interesting insights into the enzymatic properties of Ugd and Uxs. Thus, it was shown that Ugd experiences substrate inhibition, but this substrate inhibition can be relieved by the addition of ATP, despite ATP not being involved in the reaction.The primary goal of the project has been the efficient synthesis of UDPX from UDPG. To that end, the individual assay conditions have been improved to over 90% conversion efficiency for both enzymes, individually. The conversion of UDPG to UDPX is now being performed in a consecutive reaction series and the efficiency of this reaction series is being improved.Support or Funding InformationThis work was supported by NSF‐MCB grant #1121163This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.