Structural basis for substrate recognition in the Phytolacca americana glycosyltransferase PaGT3

Abstract

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.

Promiscuity and Quantitative Contribution of UGT2B17 in Drug and Steroid Metabolism Determined by Experimental and Computational Approaches

“…The highly conserved structural fold of UGT enzymes enabled the modeling of the UGT-substrate–UDP-glucuronic acid ternary complex (see the Methods Section for details). UGT2B17 shares the typical GT-B fold structure of GTs (glycosyltransferases), which consists of two Rossman-fold domains: the substrate binding domain (N-terminal) and the UDP binding domain (C-terminal) . The C-terminal and N-terminal domains are connected by a linker peptide and a long C-terminal helix.…”

“…UGT2B17 is an abundant intestinal enzyme that contributes to the variable oral PK of several newer investigational drugs such as belzutifan, 16 asciminib, 38 verinurad, 14 vorinostat, 19 and MK-7246. 14,16,18,19,28 Moreover, it plays an important role in the disposition of endogenous compounds such as testosterone and dihydrotestosterone.…”

“…UGT2B17 is an abundant intestinal enzyme that contributes to the variable oral PK of several newer investigational drugs such as belzutifan, 16 asciminib, 38 verinurad, 14 vorinostat, 19 and MK-7246. 14,16,18,19,28 Moreover, it plays an important role in the disposition of endogenous compounds such as testosterone and dihydrotestosterone. Even though the US Food and Drug Administration (FDA) recommends screening new chemical entities (NCE) for UGT metabolism, UGT2B17 is an underappreciated enzyme by pharmaceutical companies and regulatory agencies, likely because of its low expression in the liver (<3% of total UGT abundance).…”

“…15 Known functional groups prone to UGT2B17mediated glucuronidation are steroid alcohol (e.g., testosterone and androsterone), 11 nonsteroidal alcohols (e.g., belzutifan), 16 carboxylic acids (e.g., clopidogrel carboxylic acid and MK-7246), 17,18 and hydroxamic acids (e.g., vorinostat). 19 Despite these recent examples, most approved drugs and endogenous compounds that are metabolized by glucuronidation have not been evaluated as UGT2B17 substrates. The latter is likely because UGT2B17 abundance is only about 3% of the overall UGT content in the liver.…”

Supramolecular Synthons in Protein–Ligand Frameworks

“…Supramolecular chemistry, especially host–guest complexation, is contributing to solving the challenges of protein assembly and crystallization. − The protein cocrystallization capacities of synthetic macrocycles such as calixarenes, − crown ethers, − and cucurbiturils − have been amply demonstrated. In these structures, the crystal packing is often mediated by the macrocycle, in some cases to the exclusion of protein–protein interfaces. ,,, Although this work is at an early stage, there is evidence of macrocycle self-assembly that might aid protein crystal engineering.…”