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

TY - JOUR

T1 - Characterization and engineering of the bifunctional N- and O-glucosyltransferase involved in xenobiotic metabolism in plants

AU - Brazier-Hicks, Melissa

AU - Offen, Wendy A.

AU - Gershater, Markus C.

AU - Revett, Timothy J.

AU - Lim, Eng-Kiat

AU - Bowles, Dianna J.

AU - Davies, Gideon J.

AU - Edwards, Robert

PY - 2007/12/18

Y1 - 2007/12/18

N2 - 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 angstrom, 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.

AB - 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 angstrom, 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.

KW - enzymology

KW - glycosyltransferase

KW - xenobiotic

KW - glycosides

KW - domain-swapping

KW - CRYSTAL-STRUCTURE

KW - ARABIDOPSIS-THALIANA

KW - MEDICAGO-TRUNCATULA

KW - GLYCOSYLTRANSFERASES

KW - 3,4-DICHLOROANILINE

KW - REVEALS

KW - FAMILY

U2 - 10.1073/pnas.0706421104

DO - 10.1073/pnas.0706421104

M3 - Article

C2 - 18077347

SN - 0027-8424

VL - 104

SP - 20238

EP - 20243

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 51

ER -