Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, Villum Kahn Rasmussen Research Centre Pro-Active Plants, University of Copenhagen, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Plant Physiol. 2011 Jan;155(1):282-92. doi: 10.1104/pp.110.164053. Epub 2010 Nov 2.
Cassava (Manihot esculenta) is a eudicotyledonous plant that produces the valine- and isoleucine-derived cyanogenic glucosides linamarin and lotaustralin with the corresponding oximes and cyanohydrins as key intermediates. CYP79 enzymes catalyzing amino acid-to-oxime conversion in cyanogenic glucoside biosynthesis are known from several plants including cassava. The enzyme system converting oxime into cyanohydrin has previously only been identified in the monocotyledonous plant great millet (Sorghum bicolor). Using this great millet CYP71E1 sequence as a query in a Basic Local Alignment Search Tool-p search, a putative functional homolog that exhibited an approximately 50% amino acid sequence identity was found in cassava. The corresponding full-length cDNA clone was obtained from a plasmid library prepared from cassava shoot tips and was assigned CYP71E7. Heterologous expression of CYP71E7 in yeast afforded microsomes converting 2-methylpropanal oxime (valine-derived oxime) and 2-methylbutanal oxime (isoleucine-derived oxime) to the corresponding cyanohydrins, which dissociate into acetone and 2-butanone, respectively, and hydrogen cyanide. The volatile ketones were detected as 2.4-dinitrophenylhydrazone derivatives by liquid chromatography-mass spectrometry. A K(S) of approximately 0.9 μm was determined for 2-methylbutanal oxime based on substrate-binding spectra. CYP71E7 exhibits low specificity for the side chain of the substrate and catalyzes the conversion of aliphatic and aromatic oximes with turnovers of approximately 21, 17, 8, and 1 min(-1) for the oximes derived from valine, isoleucine, tyrosine, and phenylalanine, respectively. A second paralog of CYP71E7 was identified by database searches and showed approximately 90% amino acid sequence identity. In tube in situ polymerase chain reaction showed that in nearly unfolded leaves, the CYP71E7 paralogs are preferentially expressed in specific cells in the endodermis and in most cells in the first cortex cell layer. In fully unfolded leaves, the expression is pronounced in the cortex cell layer just beside the epidermis and in specific cells in the vascular tissue cortex cells. Thus, the transcripts of the CYP71E7 paralogs colocalize with CYP79D1 and CYP79D2. We conclude that CYP71E7 is the oxime-metabolizing enzyme in cyanogenic glucoside biosynthesis in cassava.
木薯（Manihot esculenta）是一种双子叶植物，它产生缬氨酸和异亮氨酸衍生的氰基葡萄糖苷亚麻苦苷和水苏碱，相应的肟和氢氰酸作为关键中间体。CYP79 酶催化氰基葡萄糖苷生物合成中的氨基酸-肟转化，已知存在于包括木薯在内的几种植物中。将肟转化为氢氰酸的酶系统以前仅在单子叶植物大刍草（高粱 bicolor）中被鉴定出来。使用这种大刍草 CYP71E1 序列作为基本局部比对搜索工具-p 搜索中的查询，在木薯中发现了一个具有大约 50%氨基酸序列同一性的假定功能同源物。从木薯茎尖制备的质粒文库中获得了相应的全长 cDNA 克隆，并将其指定为 CYP71E7。CYP71E7 在酵母中的异源表达提供了将 2-甲基丙醛肟（缬氨酸衍生的肟）和 2-甲基丁醛肟（异亮氨酸衍生的肟）转化为相应的氢氰酸的微粒体，氢氰酸分别离解为丙酮和 2-丁酮和氢氰酸。通过液相色谱-质谱法，将挥发性酮检测为 2,4-二硝基苯肼衍生物。基于底物结合谱，确定 2-甲基丁醛肟的 K(S)约为 0.9 μm。CYP71E7 对底物侧链的特异性低，催化脂肪族和芳香族肟的转化，肟分别来自缬氨酸、异亮氨酸、酪氨酸和苯丙氨酸，转化率约为 21、17、8 和 1 min(-1)。通过数据库搜索鉴定了第二个 CYP71E7 旁系同源物，其氨基酸序列约为 90%。在管内原位聚合酶链反应中，在几乎展开的叶片中，CYP71E7 旁系同源物优先在根皮层的特定细胞中和第一皮层细胞层的大多数细胞中表达。在完全展开的叶片中，在紧邻表皮的皮层细胞层和特定的维管束皮层细胞中表达明显。因此，CYP71E7 旁系同源物的转录物与 CYP79D1 和 CYP79D2 共定位。我们得出结论，CYP71E7 是木薯氰基葡萄糖苷生物合成中肟代谢酶。
