Hydrophobic recognition allows the glycosyltransferase UGT76G1 to catalyze its substrate in two orientations

Abstract

Diets high in sugar are recognized as a serious health problem, and there is a drive to reduce their consumption. Steviol glycosides are natural zero-calorie sweeteners, but the most desirable ones are biosynthesized with low yields. UGT76G1 catalyzes the β (1–3) addition of glucose to steviol glycosides, which gives them the preferred taste. UGT76G1 is able to transfer glucose to multiple steviol substrates yet remains highly specific in the glycosidic linkage it creates. Here, we report multiple complex structures of the enzyme combined with biochemical data, which reveal that the enzyme utilizes hydrophobic interactions for substrate recognition. The lack of a strict three-dimensional recognition arrangement, typical of hydrogen bonds, permits two different orientations for β (1–3) sugar addition. The use of hydrophobic recognition is unusual in a regio- and stereo-specific catalysis. Harnessing such non-specific hydrophobic interactions could have wide applications in the synthesis of complex glycoconjugates.

Crown-ether-mediated crystal structures of the glycosyltransferasePaGT3 fromPhytolacca americana

“…While the hydrophobic interactions could stabilize acceptors, the acceptors are not required to bind in a specific orientation. This is comparable with the stabilization of steviol in UGT76G1, in which hydrophobic interactions were revealed to be the main factor for binding the acceptor in two different orientations to produce two regioselective products of steviol glycosylation (Yang et al, 2019). Thus, the large hydrophobic acceptor binding pocket in PaGT3 could have resulted in the mixed glycosylated products of quercetin and resveratrol and enabled the enzyme to glycosylate both 6-hydroxyflavone and 7-hydroxyflavone efficiently.…”

“…mations for regioselective product formation, which is comparable with the acceptor recognition in UGT76G1. The hydrophobic recognition of acceptors in UGT76G1 allowed the binding of steviol in two different orientations for its regioselective glycosylation (Yang et al, 2019). For a better understanding of the acceptor binding mechanism in PaGT3, structural studies of PaGT3 with substrates are in progress.…”

“…The structure of PaGT3 also shows high similarity to those of other plant UGTs. Structural comparison of PaGT3 with the steviol-glycoside glycosyltransferase UGT76G1 (PDB entry 6inh; 28% sequence identity) from S. rebaudiana (Yang et al, 2019) and the salicylic acid glycosyltransferase UGT74F2 (PDB entry 5u6n; 28% sequence identity) from A. thaliana (George Thompson et al, 2017) indicates high structural similarity, with r.m.s.d.s of 2.2 Å for 366 C atoms and 2.3 Å for 367 C atoms, respectively. PaGT3 also shows high structural similarity to the multifunctional (iso)flavonoid glycosyltransferase UGT85H2 (PDB entry 2pq6; 27% sequence identity; Li et al, 2007) and the multifunctional triterpene/ flavonoid glycosyltransferase UGT71G1 (PDB entry 2acv; 28% sequence identity; Shao et al, 2005) from Medicago truncatula, with r.m.s.d.s of 2.1 Å for 371 C atoms and 2.2 Å for 380 C atoms, respectively.…”

“…Subsequently, many UGT structures have been determined with or without different sugar acceptors, such as PtUGT1 from Polygonum tinctorium with indoxyl sulfate (Hsu et al, 2018), UGT78K6 from Clitoria ternatea with anthocyanins (Hiromoto et al, 2015), UGT74F2 from Arabidopsis thaliana with sialic acid (George Thompson et al, 2017) and Os79 from Oryza sativa with trichothecene (Wetterhorn et al, 2016). Recently, the crystal structures of UGT76G1 from Stevia rebaudiana, which glycosylates the larger acceptor steviol (Yang et al, 2019), UGT89C1 from A. thaliana, which utilizes UDP-rhamnose as a sugar donor (Zong et al, 2019), and the C-glycosyltransferase TcCGT1 from Trollius chinensis (He et al, 2019) have been determined, which widened our knowledge of the catalytic mechanism in UGTs. However, the diversity in the polyphenols, the ability of UGTs to recognize specific compounds, the regioselectivity of products and the sugar-donor specificity of UGTs necessitates the study and determination of the crystal structures of more UGTs in order to understand the substrate recognition and the regioselectivity of the product.…”

Structure and mechanism of a redesigned multidrug transporter from the Major Facilitator Superfamily

“…1b and 4a). Most of the transporter-ligand interactions, however, are mediated through van der Waals and charge-charge interactions (for zwitterionic or cationic drugs) 22,26 , both of which lack stringent requirement for the coordination length or geometry, thereby allowing for substantial flexibility in the transporter-substrate interactions 26,44 . Indeed, previous studies supported the importance of aromatic protein residues in substrate recognition by the MFS transporter MdtM 45 , which is consistent with our observations that several aromatic amino acids are crucial for the multidrug transport mediated by the MdfA variants 26 .…”