Open AccessNon-specific activities of the major herbicide-resistance gene BAR
Author(s) -
Bastien Christ,
Ramon Hochstrasser,
Luzia Guyer,
Rita de Brito Francisco,
Sylvain Aubry,
Stefan Hörtensteiner,
JingKe Weng
Publication year - 2017
Publication title -
nature plants
Language(s) - English
Resource type - Journals
ISSN - 2055-0278
DOI - 10.1038/s41477-017-0061-1
Subject(s) - glufosinate , transgene , biology , enzyme , biochemistry , gene , amino acid , substrate (aquarium) , protein engineering , genetically modified crops , genetics , microbiology and biotechnology , ecology , glyphosate
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.