Christ B Hochstrasser R Guyer L Francisco R Aubry S Hörtensteiner S Weng JK 2017 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 1-4 . Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids 1 , indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes 5,6 . 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. 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 non-specific activities compared with its wild-type counterpart in vivo. The transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes. application/pdf https://sonar.ch/global/documents/95268 eng info:eu-repo/semantics/altIdentifier/doi/10.1038/s41477-017-0061-1 info:eu-repo/semantics/altIdentifier/pmid/29180815 info:eu-repo/semantics/openAccess Nature plants. - 2017 Acetyltransferases Arabidopsis Bacterial Proteins Brassica napus Crystallography, X-Ray Genes, Bacterial Herbicide Resistance Metabolome Models, Molecular Organophosphorus Compounds Plants, Genetically Modified Soybeans Streptomyces Triticum Non-specific activities of the major herbicide-resistance gene BAR. http://purl.org/coar/resource_type/c_6501