除草劑主要抗性基因BAR的非特異性活性

02-01

本專欄主要跟蹤各大植物學期刊最新文獻，比如Nature Plants，Molecular Plant，The Plant Cell，New Phytologist，Plant Physiology 等。
尤其以Nature Plants 為主，該期刊每一篇文獻必定跟蹤閱讀。

本文於2017年11月27日在線發表於Nature Plants，原文題目如下：

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

摘要：

雙丙氨磷抗性（BAR）和膦絲菌素乙醯轉移酶（PAT）基因通過N-乙醯化傳遞對廣譜除草劑膦絲菌素（也稱作草銨膦）的抗性，已經在基礎植物研究和基因工程作物中廣泛使用。雖然早期的體外酶測定表明，重組BAR和PAT底物對膦絲菌素具有偏好性，這種偏好性高於對20種蛋白質氨基酸的，但已經看到含BAR的轉基因在植物中的間接作用，包括氨基酸水平的修飾，但是沒有鑒定它們的直接作用。結合代謝組學，植物遺傳學和生物化學方法，結果顯示，在幾種植物物種中，轉基因BAR確實將兩種植物內源氨基酸氨基，己二酸和色氨酸，轉化為它們各自的N-乙醯化產物。我們報道了BAR的晶體結構，並進一步描述了其底物選擇性和催化機理的結構基礎。通過結構引導的蛋白質工程，我們產生了幾個BAR變體，與其野生型對應物相比，在體內顯示出顯著減少的非特異性活性。酶的轉基因表達可導致由酶混亂引起的隨意的脫靶代謝。在機械水平上理解這種現象，可以以異源表達的酶為特徵，最大限度的促進絕緣系統的設計。

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. 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.

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