Dataset Information

Nonspecific activities of the major herbicide-resistance gene BAR

ABSTRACT: Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops. Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids, indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes. Combining metabolomics, plant genetics, and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species examined. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced nonspecific activities compared to its wild-type counterpart in vivo. Our results demonstrate that transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding of such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes.

INSTRUMENT(S): Exactive (Thermo Scientific), TSQ Quantum Access MAX (Thermo Scientific), QqTOF Compact (Bruker)

SUBMITTER: Bastien Christ

PROVIDER: MTBLS553 | MetaboLights | 2017-12-05

REPOSITORIES: MetaboLights

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Non-specific activities of the major herbicide-resistance gene BAR.

Christ Bastien B Hochstrasser Ramon R Guyer Luzia L Francisco Rita R Aubry Sylvain S Hörtensteiner Stefan S Weng Jing-Ke JK

Nature plants 20171127 12

Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops <sup>1-4</sup> . Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids <sup>1</sup> , indirect effects of BAR ...[more]

PMID: 29180815

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