Purification and cloning of an esterase from the weed black-grass (Alopecurus myosuroides), which bioactivates aryloxyphenoxypropionate herbicides
Purification and cloning of an esterase from the weed black-grass (Alopecurus myosuroides), which bioactivates aryloxyphenoxypropionate herbicides
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DOI：
10.1111/j.1365-313x.2004.02174.x
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发表时间：
2004-09-01
期刊：
影响因子：
7.2
通讯作者：
Edwards, R
中科院分区：
文献类型：
--
作者：
Cummins, I;Edwards, R
在北欧谷物作物的问题杂草黑草（Alopecurus myosuroides）中发现了通过水解酯前体使芳基苯氧丙酸酯（AOPP）杀菌剂产生生物活性除草酸的羧酶。优势的40 kDa羧酶被纯化了1700倍，并通过生物素化氟膦酸自杀底物亲和标记鉴定为丝氨酸水解酶。MS-MS测序的肽消化鉴定它是GDSL家族丝氨酸水解酶的一员。利用RACE-PCR技术克隆了全长a . myosuroides水解酶（Amgdsh1），并在酵母毕赤酵母中作为分泌酶表达。表达与AOPP酯活性相关。预计AmGDSH1将被糖基化并输出到植物的外质体。通过对单子叶植物中相关序列的分析，提出了GDSL植物水解酶超家族的另一种分类方法，并讨论了其在作物和杂草内源代谢和除草剂生物活化中的重要性。
Carboxyesterases which activate aryloxyphenoxypropionate (AOPP) graminicides to their bioactive herbicidal acids by hydrolysing the respective ester precursors have been identified in black-grass (Alopecurus myosuroides), a problem weed of cereal crops in Northern Europe. The dominant 40 kDa carboxyesterase was purified 1700-fold and identified as a serine hydrolase by affinity labelling with a biotinylated fluorophosphonate suicide substrate. MS-MS sequencing of a peptide digest identified it to be a member of the GDSL family of serine hydrolases. The full-length A. myosuroides hydrolase (Amgdsh1) was cloned by RACE-PCR and expressed in the yeast Pichia pastoris as a secreted enzyme. Expression was associated with activity towards AOPP esters. AmGDSH1 was predicted to be glycosylated and exported to the apoplast in planta. Based on the analysis of related sequences in monocotyledonous plants an alternative classification of the GDSL plant hydrolase superfamily is suggested and their importance in endogenous metabolism and herbicide bioactivation in crops and weeds discussed.