Differential hydrolysis of the flavonoid hesperidin by Actinoplanes missouriensis 431T glycosidases

BAGLIONI, MICAELA; FRIES A; MULLER M; BRECCIA JD; MAZZAFERRO LS

Congreso; BIOTRANS 2021; 2021

Actinoplanes missouriensis 431T is a soil isolate bacterium, well known for its ability to degrade flavonoids. Using an activity-based screening in combination with a sequence analysis approach, two enzymes active on the flavonoid hesperidin (hesperitin-7-O-β-rutinoside) were discovered and recombinantly expressed: the diglycosidase hesperidin 6-O-α-l-rhamnosyl-β-d-glucosidase (αRβG, EC 3.2.1.168) and a α-l-rhamnosidase (αR, EC 3.2.1.40) (Figure 1)[1,2]. While αRβG hydrolyzes hesperidin at the heterosidic bond releasing the disaccharide rutinose (6-O-α-l--rhamnosyl-d-glucose) and the aglycone hesperetin, aR recognizes the glycosidic bond between the rhamnose and glucose moieties, releasing rhamnose and glucosylated hesperetin (Figure 1). In both cases, the enzymes obeyed typical Michaelis-Menten kinetics with hesperidin as the substrate (αRβG: KM 1.6±0.5 mM, Vmax 0.0013±0.0002 μmol min-1; αR: KM 0.8 ± 0.2 mM, Vmax 0.061±0.003 μmol min-1). Regarding substrate recognition, the αRβG showed specificity for 7-O-rutinosylated flavonoids and only trace of activity with the monoglycoconjugate p-nitrophenyl β-d-glucopyranoside. In contrast, the αR exhibited higher promiscuity, with activity against the disaccharide rutinose, 7-O-rutinosyl-flavanones, 3-O-rutinosyl-flavonols, and rhamnose containing polymers. Actinoplanes missouriensis 431T is a soil isolate bacterium, well known for its ability to degrade flavonoids. Using an activity-based screening in combination with a sequence analysis approach, two enzymes active on the flavonoid hesperidin (hesperitin-7-O-β-rutinoside) were discovered and recombinantly expressed: the diglycosidase hesperidin 6-O-α-l-rhamnosyl-β-d-glucosidase (αRβG, EC 3.2.1.168) and a α-rhamnosidase (αR, EC 3.2.1.40) (Figure 1)[1,2]. While αRβG hydrolyzes hesperidin at the heterosidic bond releasing the disaccharide rutinose (6-O-α-l-rhamnosyld-glucose) and the aglycone hesperetin, aR recognizes the glycosidic bond between the rhamnose and glucose moieties, releasing rhamnose and glucosylated hesperetin (Figure 1). In both cases, the enzymes obeyed typical Michaelis-Menten kinetics with hesperidin as the substrate (αRβG: KM 1.6±0.5 mM, Vmax 0.0013±0.0002 μmol min-1; αR: KM 0.8 ± 0.2 mM, Vmax 0.061±0.003 μmol min-1). Regarding substrate recognition, the αRβG showed specificity for 7-O-rutinosylated flavonoids and only trace of activity with the monoglycoconjugate p-nitrophenyl β-d-glucopyranoside. In contrast, the αR exhibited higher promiscuity, with activity against the disaccharide rutinose, 7-O-rutinosyl-flavanones, 3-O-rutinosyl-flavonols, and rhamnose containing polymers.