Liverworts are rich in bibenzyls and related O-glycosides, which show antioxidant activity. However, glycosyltransferases that catalyze the glycosylation of bibenzyls have not yet been characterized. Here, we identified two bibenzyl UDP-glucosyltransferases named MpUGT737B1 and MpUGT741A1 from the model liverwort Marchantia polymorpha. The in vitro enzymatic assay revealed that MpUGT741A1 specifically accepted the bibenzyl lunularin as substrate. MpUGT737B1 could accept bibenzyls, dihydrochalcone and phenylpropanoids as substrates, and could convert phloretin to phloretin-4-O-glucoside and phloridzin, which showed inhibitory activity against tyrosinase and antioxidant activity. The results of sugar donor selectivity showed that MpUGT737B1 and MpUGT741A1 could only accept UDP-glucose as a substrate. The expression levels of these MpUGTs were considerably increased after UV irradiation, which generally caused oxidative damage. This result indicates that MpUGT737B1 and MpUGT741A1 may play a role in plant stress adaption. Subcellular localization indicates that MpUGT737B1 and MpUGT741A1 were expressed in the cytoplasm and nucleus. These enzymes should provide candidate genes for the synthesis of bioactive bibenzyl O-glucosides and the improvement of plant antioxidant capacity.
Synthetic efforts of homogeneous heparin oligosaccharides are increasing in terms of the number of products and the synthetic scale. The dual-function glycosyltransferase Pasteurella multocida heparosan synthase 2 (PmHS2) is widely...
Glycosaminoglycan synthases have
immense potential in
applications
involving synthesis of oligosaccharides, using enzymatic approaches
and construction of cell factories that produce polysaccharides as
critical metabolic components. However, the use of high-throughput
activity assays to screen for the evolution of these enzymes can be
challenging because there are no significant changes in fluorescence
or absorbance associated with glycosidic bond formation. Here, using
incorporation of azido-labeled N-acetylhexosamine
analogs into bacterial capsule polysaccharides via bacterial metabolism
and bioorthogonal chemistry, fluorophores were specifically introduced
onto cell surfaces. Furthermore, correlations between detectable fluorescence
signals and the polysaccharide-synthesizing capacity of individual
bacteria were established. Among 10 candidate genes, 6 members of
the chondroitin synthase family were quickly identified in a recombinant Bacillus subtilis host strain. Additionally, directed
evolution of heparosan synthase was successfully performed using fluorescence-activated
cell sorting of recombinant Escherichia coli O10:K5(L):H4, yielding several mutants with increased activity.
Cell-based approaches that selectively detect the presence or absence
of synthases within an individual colony of bacterial cells, as well
as their level of activity, have broad potential in the exploration
and engineering of glycosaminoglycan synthases. These approaches also
support the creation of novel strategies for high-throughput screening
of enzyme activity based on cell systems.
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