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 N-glucosylating 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 A, 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.

Fig. 1.

Metabolic fate of pesticides and pollutants in plants, showing the central importance of N- and O-glycosylation. (A) After either direct uptake, or after phase 1 metabolism, xenobiotics bearing amine (R-NH₂) or hydroxyl (R-OH) groups undergo phase 2 conjugation with glucose (G), as catalyzed by UGTs. Conjugates may then be exported from the cell and hydrolyzed to release the aglycon or imported (phase 3) into the vacuole. As an alternative to phase 2 glycosylation, xenobiotic metabolites can also undergo phase 4 polymerization into cell wall components to form insoluble bound residues. (B) Reactions catalyzed by UGT72B1; the glucosylation of phenols (X = O) and anilines (X = NH) with inversion of anomeric configuration to generate the β-D glucosides as products.

Fig. 2.

Screening of the type 1 UGT superfamily of Arabidopsis for O-, N-, and S-conjugating activity. (A) The phylogenetic tree of the Arabidopsis UGTs showing the family members that did (black) and did not (red) express as GST-fusion proteins when expressed in E. coli in the pGEX vector. Enzymes showing activity toward xenobiotic substrates are shown in blue. (B) The acceptors used for determining OGT (TCP), NGT (DCA), and SGT (CTP) activity.

Fig. 3.

Structure of UGT72B1. (A) 3D structure of UGT72B1 as a protein diagram “color-ramped” from the N (blue) to the C terminus (red). (B) Observed electron density (2F_obs − F_calc, α_calc) for 2,4,5-TCP and UDP-2FGlc at the active center of UGT72B1 with residues discussed in the text, shown in ball and stick. (C) Overlay of the 2,4,5-TCP/UDP-2FGlc complex of UGT72B1 (this work) with the kaempferol/UDP-2FGlc complex of V. vinifera UGT1 (12) (UDP truncated at the β-phosphate), illustrating the differing geometric disposition of His and Asp between the two enzymes, contributed in part by the environment of the histidines. (D) Partial sequence alignment of the BnUGT and UGT72B1 showing the region implicated in N vs. O specificity. The structures were drawn by using MOLSCRIPT (30) and BOBSCRIPT (31).