Active site cleft mutants of Os9BGlu31 transglucosidase modify acceptor substrate specificity and allow production of multiple kaempferol glycosides.

BACKGROUND

Rice Os9BGlu31 is a transglucosidase that can transfer glucose to phenolic acids, flavonoids, and phytohormones. Os9BGlu31 displays a broad specificity with phenolic 1-O-β-D-glucose esters acting as better glucose donors than glucosides, whereas the free phenolic acids of these esters are also excellent acceptor substrates.

METHODS

Based on homology modeling of this enzyme, we made single point mutations of residues surrounding the acceptor binding region of the Os9BGlu31 active site. Products of the wild type and mutant enzymes in transglycosylation of phenolic acceptors from 4-nitrophenyl β-D-glucopyranoside donor were identified and measured by UPLC and negative ion electrospray ionization tandem mass spectrometry (LCMSMS).

RESULTS

The most active variant produced was W243N, while I172T and L183Q mutations decreased the activity, and other mutations at W243 (A, D, M, N, F and Y) had variable effects, depending on the acceptor substrate. The Os9BGlu31 W243N mutant activity was higher than that of wild type on phenolic acids and kaempferol, a flavonol containing 4 hydroxyl groups, and the wild type Os9BGlu31 produced only a single product from each of these acceptors in significant amounts, while W243 variants produced multiple glucoconjugates. Fragmentation analysis provisionally identified the kaempferol transglycosylation products as kaempferol 3-O, 7-O, and 4'-O glucosides and 3,7-O, 4',7-O, and 3,4'-O bis-O-glucosides. The Os9BGlu31 W243 mutants were also better able to use kaempferol 3-O-glucoside as a donor substrate.

GENERAL SIGNIFICANCE

The W243 residue was found to be critical to the substrate and product specificity of Os9BGlu31 transglucosidase and mutation of this residue allows production of a range of glucoconjugates.