Structural and functional study of uridine diphosphate glycosyltransferases from Phytolacca americana

概要

Polyphenols are natural products with several health benefits and serve as an important lead compound for drug discovery. These compounds have gained significant interest in pharmaceutical, cosmetics, and food industries since they are effective against cancer, diabetes, inflammation, cardiovascular diseases, neurodegenerative diseases, UV radiation protection, aging, and obesity. However, the poor water solubility and stability of polyphenols, which results in low bioavailability, are limiting their exploitation. Glycosylation is one of the important methods to improve the solubility and stability of polyphenols. In plants, uridine diphosphate glycosyltransferases (UGTs) catalyze glycosylation of small molecules. Glycosylation is important for the cellular localization, storage, and metabolism of such compounds. Thus, understanding the structure and glycosylation mechanism of UGTs will be useful for improving the solubility and stability of compounds with pharmaceutical interest.

Here, two glycosyltransferases, PaGT2 and PaGT3, from a plant Phytolacca americana,“Yoshuyamagobou” in Japanese, were studied. Among the three glycosyltransferases (PaGT1/2/3) identified in P. americana, PaGT2 had the least and PaGT3 had the highest glycosylation activities with different phenolic compounds. Understanding the reasons for this discrepancy will add information to the substrate recognition and glycosylation mechanisms of UGTs, which will be useful for developing efficient enzymes with higher substrate specificity. For this purpose, the crystal structures of PaGT2 and PaGT3 with and without substrates were determined. Molecular docking, site-directed mutagenesis, and glycosylation assays were also performed. The information was used to modify PaGT2 for resveratrol glycosylation, which was negligibly glycosylated by wildtype PaGT2.

In chapter 1, the crystal structures of PaGT2 with and without substrates were determined.PaGT2 has a very low activity with resveratrol and pterostilbene that are present in the crystal structures. The crystal structures show that the OH groups on these substrates are not close enough to the conserved catalytic base (His18) for effective interactions and catalysis. As PaGT2 does not glycosylate the substrates in the crystal structures, molecular docking with good substrates piceatannol and kaempferol is performed in the structure of PaGT2. Besides the conserved catalytic pair (His18- Asp119), both the crystal structures and molecular docking suggests His81 as an additional catalytic residue in PaGT2. The catalytic roles of His18 and His81 are studied by site-directed mutagenesis and glycosylation assays. The structural and functional studies identified two separate catalytic residues in a single UGT for the first time.

In chapter 2, PaGT2 was modified to enable resveratrol glycosylation. Resveratrol is one of the most studied stilbenoid polyphenols for its beneficial bioactivities. In the crystal structure, the OH groups on resveratrol are far from both catalytic bases. It is assumed that changing the binding position of resveratrol could enable its glycosylation by PaGT2. A residue in the active-site that could affect the binding of resveratrol is identified as Cys142. Cys142 is mutated to glycine, alanine,phenylalanine, and glutamine. The mutant enzymes transform resveratrol into resveratrol glucosides. The crystal structure of the Cys142Phe mutant shows the substitution of Cys142 could alter the position of resveratrol in the active-site than in the wildtype enzyme. Besides, the crystal structure of the Cys142Ala mutant is determined with UDP, which reveals that His81 could inhibit glycosylation, despite being a complementary catalytic residue. This is also verified by the measurement of resveratrol glycosylation activity using the His18Ala/Cys142Ala and the His81Ala/Cys142Ala double mutants. The His81Ala/Cys142Ala double mutant has higher activity and regioselectivity for resveratrol glycosylation than both the Cys142Ala and the His18Ala/Cys142Ala double mutants.

In chapter 3, the crystal structures of PaGT3 with and without substrates were discussed.Although PaGT3 glycosylates a range of compounds, the glycoside products of smaller compounds have low regioselectivity. The crystal structures showed PaGT3 has a large, hydrophobic, and flexible acceptor binding pocket. In the kaempferol complexed structure, the substrate positions in two different orientations in two molecules of PaGT3 present in the asymmetric unit. Besides kaempferol, the acceptor- binding pocket also contains some ethylene glycol molecules. This suggests the large acceptor-binding pocket in PaGT3 allows smaller polyphenols to bind in different orientations, which results in low product regioselectivity. The PaGT3 structure with capsaicin shows that the acceptor binding pocket in PaGT3 easily accommodates long-chain phenolic compounds. The elongated acceptor binding pocket in PaGT3 is an important feature that allows it to accept and glycosylate long-chain phenolic compounds such as capsaicin and retinol. Besides, crown ether was used as an additive for the crystallization of PaGT3. Although crown ethers have been used in protein crystallization, the structures of PaGT3 reveals a new role of these compounds in protein crystallization. In the structures of PaGT3, crown ether-metal complex acts as molecular glue for the crystal growth.

The structural and functional studies of PaGT2 identified two separate catalytic residues in the enzyme wherein one of the catalytic residue (His18) is conserved among UGTs and the other (His81) is not. The study finds His18 is the primary residue for catalysis. Although His81 in PaGT2 catalyzes glycosylation, it also partly inhibits the enzyme. The crystal structure of the Cys142Phe mutant shows that the mutation of Cys142 changes the binding position of resveratrol in the active-site which allows the Cys142 mutant enzymes to glycosylate the compound. Mutation of both His81 and Cys142 significantly improved the enzyme’s glycosylation activity towards resveratrol. The crystal structure of PaGT3 shows that the acceptors in the enzyme’s active-site are stabilized mainly through hydrophobic interactions. The stabilization of substrates through hydrophobic interactions does not require their specific orientation. Thus, the product regioselectivity is lower in PaGT3 although it has a higher glycosylation activity compared to PaGT2. The larger hydrophobic acceptor binding pocket in PaGT3 also allows it to recognize long-chain phenolic compounds such as capsaicin and retinol, which are not glycosylated by PaGT2. The findings in this study provide information for the customization of UGTs with high substrate specificity and product regioselectivity for glycosylation of compounds of pharmaceutical, nutraceutical, and cosmetics interests.