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Optimization of the Paired Enzyme Assay Synthesizing UDP‐Xylose from UDP‐Glucose
Author(s) -
Cook Matthew D,
Culbertson Alan,
Zabotina Olga
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.764.16
Subject(s) - enzyme , chemistry , xylose , biochemistry , substrate (aquarium) , nad+ kinase , dehydrogenase , chromatography , biology , fermentation , ecology
UDP‐xylose (UDPX) is the substrate used for xylosylation in most organisms. Xylosyl residues are components of vital molecules in the cell walls of plants and bacteria and in the extracellular matrices of animals. Despite the proliferation of xylosylated compounds, UDPX is a markedly expensive chemical which limits research potential into these areas. It is known that the pair of enzymes UDP‐glucose dehydrogenase (UGD) and UDPX synthase (UXS) can catalyze the set of reactions leading from UDP‐glucose, a much cheaper chemical, to UDPX. This project optimized the enzyme assay of both UGD and UXS to improve the efficiency and rate of UDPX synthesis in order to develop an economical means of producing UDPX. Additionally, the kinetics of the previously uncharacterized UGD and UXS isoforms used in the assay were investigated. Enzyme assays were performed, systematically comparing the rates of reaction and final UDPX‐production efficiency under different assay compositions and component concentrations for both enzymes individually and then together. The UGD assay was optimized first. As a result of reacting UDPG to the product UDP‐glucuronic acid, two equivalents of NAD + were reduced to NADH. The generation of NADH could be analyzed spectrophotometrically at a 340 nm wavelength. Results from optimization have assay efficiency approaching 100% and the rate of reaction has improved appreciably. The earliest assays reacted with a maximum rate of 1.4 × 10 −5 umol UDPG/min/ug UGD. Optimized assays have improved to 1.0 × 10 −3 umol UDPG/min/ug UGD, a nearly 100‐fold improvement. Work on UXS proceeds. Support or Funding Information NSF‐MCB Grant # 1121163