The
glycosylation of small hydrophobic compounds is catalyzed by
uridine diphosphate glycosyltransferases (UGTs). Because glycosylation
is an invaluable tool for improving the stability and water solubility
of hydrophobic compounds, UGTs have attracted attention for their
application in the food, cosmetics, and pharmaceutical industries.
However, the ability of UGTs to accept and glycosylate a wide range
of substrates is not clearly understood due to the existence of a
large number of UGTs. PaGT2, a UGT from Phytolacca
americana, can regioselectively glycosylate piceatannol but
has low activity toward other stilbenoids. To elucidate the substrate
specificity and catalytic mechanism, we determined the crystal structures
of PaGT2 with and without substrates and performed
molecular docking studies. The structures have revealed key residues
involved in substrate recognition and suggest the presence of a nonconserved
catalytic residue (His81) in addition to the highly conserved catalytic
histidine in UGTs (His18). The role of the identified residues in
substrate recognition and catalysis is elucidated with the mutational
assay. Additionally, the structure-guided mutation of Cys142 to other
residues, Ala, Phe, and Gln, allows PaGT2 to glycosylate
resveratrol with high regioselectivity, which is negligibly glycosylated
by the wild-type enzyme. These results provide a basis for tailoring
an efficient glycosyltransferase.
This study was supported by funding from the National Institute for Public Health and the Environment, The Netherlands; Oxford Vaccine Group, University of Oxford, UK; and GlaxoSmithKline Biologicals, Belgium.
Uridine diphosphate glycosyltransferases (UGTs) are ubiquitous enzymes that are involved in the glycosylation of small molecules. As glycosylation improves the water solubility and stability of hydrophobic compounds, interest in the use of UGTs for the synthesis of glycosides of poorly soluble compounds is increasing. While sugar-donor recognition in UGTs is conserved with the presence of a plant secondary product glycosyltransferase (PSPG) motif, the basis of the recognition of the sugar acceptor and the regioselectivity of the products is poorly understood owing to low sequence identity around the acceptor-binding region. PaGT3, a glycosyltransferase from the plant Phytolacca americana, can glycosylate a range of acceptors. To illustrate the structurefunction relationship of PaGT3, its crystal structure was determined. The sugardonor and sugar-acceptor binding pockets in PaGT3 were recognized by comparison of its structure with those of other UGTs. The key feature of PaGT3 was the presence of longer loop regions around the hydrophobic acceptorbinding pocket, which resulted in a flexible and wider acceptor binding pocket. In this study, PaGT3 crystals were grown by co-crystallization with 18-crown-6 ether or 15-crown-5 ether. The crown-ether molecule in the asymmetric unit was observed to form a complex with a metal ion, which was coordinated on two sides by the main-chain O atoms of Glu238 from two molecules of the protein.The crown ether-metal complex resembles a molecular glue that sticks two molecules of PaGT3 together to enhance crystal growth. Thus, this result provides an insight into the substrate-recognition strategy in PaGT3 for the study of glycosyltransferases. Additionally, it is shown that crown ether-metal ion complexes can be used as a molecular glue for the crystallization of proteins.
Capsaicinoids are phenolic compounds that have health benefits. However, the pungency and poor water solubility of these compounds limit their exploitation. Glycosylation is a powerful method to improve water solubility and reduce pungency while preserving bioactivity. PaGT3, a uridine diphosphate glycosyltransferase (UGT) from Phytolacca americana, is known for its ability to glycosylate capsaicinoids and other phenolic compounds. While structural information on several UGTs is available, structures of UGTs that can glycosylate a range of phenolic compounds are rare. To fill this gap, crystal structures of PaGT3 with a sugar-donor analogue (UDP-2-fluoroglucose) and the acceptors capsaicin and kaempferol were determined. PaGT3 adopts a GT-B-fold structure that is highly conserved among UGTs. However, the acceptor-binding pocket in PaGT3 is hydrophobic and large, and is surrounded by longer loops. The larger acceptor-binding pocket in PaGT3 allows the enzyme to bind a range of compounds, while the flexibility of the longer loops possibly plays a role in accommodating the acceptors in the binding pocket according to their shape and size. This structural information provides insights into the acceptor-binding mechanism in UGTs that bind multiple substrates.
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