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.

中文翻译：

---

主要除草剂抗性基因BAR的非特异性活性。

通过N-乙酰化传达对广谱除草剂膦丝菌素（也称为草铵膦）的抗性的双丙氨膦抗性（BAR）和草丁膦乙酰基转移酶（PAT）基因已在全球基础植物研究和转基因作物^{1-4中广泛使用}。尽管早期的体外酶分析显示重组BAR和PAT在20个蛋白原氨基酸^1上显示出对膦丝菌素的底物优先性，但已观察到植物中含有BAR的转基因的间接作用，包括修饰的氨基酸水平，但尚未鉴定它们的作用。直接原因^5,6。结合代谢组学，植物遗传学和生物化学方法，我们表明转基因BAR确实将几种植物物种中的两种植物内源氨基酸，氨基己二酸和色氨酸转化为它们各自的N-乙酰化产物。我们报告了BAR的晶体结构，并进一步描述了其底物选择性和催化机理的结构基础。通过结构指导的蛋白质工程，我们产生了几种BAR变体，与野生型对应的活体相比，它们显示出显着降低的非特异性活性。酶的转基因表达可导致因酶混杂而引起的意外脱靶代谢。在机械水平上理解此类现象可有助于设计具有异源表达酶的最大绝缘系统。