Abstract
Uridine diphosphate galactose (UDP-galactose) is a valuable building block in the enzymatic synthesis of galactose-containing glycoconjugates. UDP-glucose 4-epimerase (UGE) is an enzyme which catalyzes the reversible conversion of abundantly available UDP-glucose to UDP-galactose. Herein, we described the cloning, expression, purification, and biochemical characterization of an unstudied UGE from the oyster Magallana gigas (MgUGE). Activity tests of recombinantly expressed MgUGE, using HPLC (high-performance liquid chromatography), mass spectrometry, and photometric assays, showed an optimal temperature of 16 °C, and reasonable thermal stability up to 37 °C. No metal ions were required for enzymatic activity. The simple nickel-affinity-purification procedure makes MgUGE a valuable biocatalyst for the synthesis of UDP-galactose from UDP-glucose. The biosynthetic potential of MgUGE was further exemplified in a coupled enzymatic reaction with an oyster-derived β-1,4-galactosyltransferase (MgGalT7), allowing the galactosylation of the model substrate para-nitrophenol xylose (pNP-xylose) using UDP-glucose as the starting material.
Highlights
Galactose-containing carbohydrates are widely distributed in living systems, and play important biological roles as structural elements, energy sources, and precursors for cell surface elements
MgUGE was fully inactivated by the addition of low concentrations (0.1% w/v) of sodium dodecyl sulfate (SDS)
The activity test relied on the activity of MgUGE to convert UDP-galactose to UDP-glucose, which was subsequently oxidized in the presence of NAD+ by Sphaerobacter thermophilus UDP-glucose dehydrogenase (StUGD), forming UDP-glucuronic acid and NADH [32] (Figure 3)
Summary
Galactose-containing carbohydrates are widely distributed in living systems, and play important biological roles as structural elements, energy sources, and precursors for cell surface elements. Galactose activation, through which galactose is converted into its active form uridine diphosphate galactose (UDP-galactose), is essential prior to the formation of galactose-modified conjugates. This procedure is catalyzed by the enzyme UDP-glucose 4-epimerase (UGE). CClloonniinngg aanndd HHoommoollooggyy AAnnaallyyssiiss ooff tthhee OOyysstteerr UUGGEE GGeennee. The Km value of MgUGE was 1.6 ± 0.1 mM for UDP-galactose, the νmax value was 270 ± 9 μM∙min−1, and the kcat value was calculated to be 134 ± 12 min−1 (Figure S4). 1T,haendKmthveaklcuate of MgUGE was 1.6 ± 0.1 mM for UDP-galactose, the νmax value was calculated to be 134 ± 12 min−1 (Figure S4). TvhaelupesHovf athlueeTsroisf-tHheClTarinsd-HsCodl iaunmd spohdoisupmhapteh-ocsitprhataeteb-uciftfreartserbeupfrfeesresnrtetphreeisrepnHt tvheailruepsHatv2a2lu◦eCs.aTth2e2e°rCro. rTbhaerserrreoprrebsaernstrtehperesstaenndt athrde sdteavnidaatirodnsdecvalicautiloantesdcafrlocumlattherdeefrionmdetpherneedeinndt eepxpenedriemnetnetxsp. eriments
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