Characterization and engineering of the bifunctional N - and O -glucosyltransferase involved in xenobiotic metabolism in plants

Abstract

The glucosylation of pollutant and pesticide metabolites in plants controls their bioactivity and the formation of subsequent chemical residues. The model plant Arabidopsis thaliana contains >100 glycosyltransferases (GTs) dedicated to small-molecule conjugation and, whereas 44 of these enzymes catalyze the O-glucosylation of chlorinated phenols, only one, UGT72B1, shows appreciable Nglucosylating activity toward chloroanilines. UGT72B1 is a bifunctional O-glucosyltransferase (OGT) and N-glucosyltransferase (NGT). To investigate this unique dual activity, the structure of the protein was solved, at resolutions up to 1.45 Å, in various forms including the Michaelis complex with intact donor analog and trichlorophenol acceptor. The catalytic mechanism and basis for O/N specificity was probed by mutagenesis and domain shuffling with an orthologous enzyme from Brassica napus (BnUGT), which possesses only OGT activity. Mutation of BnUGT at just two positions (D312N and F315Y) installed high levels of NGT activity. Molecular modeling revealed the connectivity of these residues to H19 on UGT72B1, with its mutagenesis exclusively defining NGT activity in the Arabidopsis enzyme. These results shed light on the conjugation of nonnatural substrates by plant GTs, highlighting the catalytic plasticity of this enzyme class and the ability to engineer unusual and desirable transfer to nitrogen-based acceptors.enzymology ͉ glycosyltransferase ͉ xenobiotic ͉ glycosides ͉ domain-swapping P lants are constantly exposed to synthetic compounds, such as pollutants and crop protection agents, and are able to transform these xenobiotics by using a four-phase detoxification system that has immediate parallels with drug metabolism in animals (Fig. 1A). Absorbed xenobiotics are first metabolically activated by ''phase 1'' enzymes, which then facilitates their subsequent bioconjugation with polar natural products (amino acids, sugars, peptides) in phase 2 metabolism. In crops and weeds, the most commonly observed phase 2 reaction is glycosylation (1), a reaction catalyzed by family GT1 glycosyltransferases (2), which are more normally engaged in secondary metabolism (3). A diverse range of xenobiotics are known to undergo conjugation as O-, S-, and N-acceptors, with UDP-glucose (UDP-glc) being the most commonly observed sugar donor (1). Once synthesized, conjugates accumulate transiently in the cytosol before being transported (phase 3) to either the vacuole or apoplast (Fig. 1 A).Despite their central importance in the metabolism of herbicides, pesticides, and organic pollutants, the identity of the enzymes catalyzing the glycosylation of xenobiotics has only recently been determined through studying their activity in the model plant Arabidopsis thaliana (4,5). Arabidopsis plants rapidly metabolize persistent pollutants such as 2,4,5-trichlorophenol (TCP) and 3,4-dichloroaniline (DCA) by O-and N-glucosylation, respectively (4-7) (Fig. 1B). Several UDP-glc-dependent glycosyltransferases (UGTs) in Arabidopsis have been shown to have O-gluco...

The C-Glycosylation of Flavonoids in Cereals

“…The most similar proteins in Arabidopsis thaliana (27-29% identity) are group E UGTs that catalyze the O-glucosylation of monolignols (38,39). In addition, the group E member UGT72B1 is able to both O-glucosylate and N-glucosylate xenobiotic pollutants (26). None of the group E UGTs are known to C-glucosylate acceptors, with UGT72B1 showing no activity toward the 2-hydroxyflavanone substrates in our hands (data not shown).…”

“…The program MODELLER (25) was subsequently used to build a homology model based on the crystal structure of UGT72B1 (26). Model scores of 1 and the combined model quality score of 1.36 indicated reliability in the model as expected with a sequence identity of 35% (27).…”

A transcript profiling approach reveals an epicatechin-specific glucosyltransferase expressed in the seed coat of Medicago truncatula

“…Both are multifunctional. UGT72B1 catalyzed both N-and O-glycosylation of xenobiotics (31), and AS glucosylated 45 out of 74 potential phenolic acceptors (32), in remarkable contrast to the apparently strict substrate specificity of UGT72L1.…”

Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L.

“…Besides the arbutin synthase from R. serpentina (Hefner et al 2002) mentioned above, subfamily 72 contains A. thaliana glucosyltransferases, including UGT72B1, which possesses both N-and O-glucosyltransferase activity. This enzyme was assigned to the detoxiWcation of xenobiotics (Loutre et al 2003;Brazier-Hicks and Edwards 2005;Brazier-Hicks et al 2007), whereas the UGTs 72E1-E3 are involved in the glucosylation of monolignols, hydroxycinnamic acids, and hydroxycinnamic aldehydes Lanot et al 2006). H. pilosella UGT90A7 belongs to group C and, to the best of our knowledge, an enzyme of this subfamily has never been characterised.…”