RT Journal Article SR Electronic T1 Sucrose phosphorylase from Alteromonas mediterranea: structural insight into the regioselective α-glucosylation of (+)-catechin JF bioRxiv FD Cold Spring Harbor Laboratory SP 2023.04.11.536264 DO 10.1101/2023.04.11.536264 A1 Marine Goux A1 Marie Demonceaux A1 Johann Hendrickx A1 Claude Solleux A1 Emilie Lormeau A1 Folmer Fredslund A1 David Tezé A1 Bernard Offmann A1 Corinne André-Miral YR 2023 UL http://biorxiv.org/content/early/2023/04/11/2023.04.11.536264.abstract AB Flavonoids glycosylation at different positions is paramount to solubility and modulation of bioactivities. Sucrose phosphorylases, through transglycosylation reactions, are interesting enzymes that can transfer glucose from sucrose, the donor substrate, onto polyphenols to form glycoconjugates. Here, we report for the first time the structural and enzymatic properties of sucrose phosphorylase from the marine bacteria Alteromonas mediterranea (AmSP). We characterized and investigated the transglucosylation capacity of two new variants of the enzyme on (+)-catechin and their propensity to catalyse its regioselective glucosylation. AmSP-Q353F and AmSP-P140D were shown to catalyse the regiospecific glucosylation of (+)-catechin using sucrose as donor substrate. While AmSP-WT was devoid of synthetic activity, each of its two single mutant provided high yields of specific regioisomers: 89% of (+)-catechin-4′-O-α-D-glucopyranoside (CAT-4’) for AmSP-P140D and 92% of (+)-catechin-3′-O-α-D-glucopyranoside (CAT-3’) for AmSP-Q353F. The novel compound CAT-4’ was fully characterized by NMR and mass spectrometry. We used molecular docking simulations on structural models of the glucosyl-enzyme intermediate to explain this regioselectivity. We showed that AmSP-P140D preferentially binds (+)-catechin in a mode that favours glucosylation on its hydroxyl group in position 4’ (OH-4’) while the binding mode of the flavonoid in AmSP-Q353F favoured glucosylation on its hydroxyl group in position 3’ (OH-3’).Competing Interest StatementThe authors have declared no competing interest.