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

MICAELA BAGLIONI; ALEXANDER FRIES; MICHAEL MULLER; JAVIER BRECCIA; LAURA S. MAZZAFERRO

Graz

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.The use of the glycosidases with different specificities represents an interesting strategy for the structural diversification of plant secondary metabolites. The enzymes αRβG and αR from A. missouriensis 431T are thus a contribution to the toolkit of carbohydrate-active enzymes for biocatalytic applications.