| Title: | "Cytochrome P450 CYP71AT96 catalyses the final step of herbivore-induced phenylacetonitrile biosynthesis in the giant knotweed, Fallopia sachalinensis" |
| Author(s): | Yamaguchi T; Noge K; Asano Y; |
| Address: | "Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan. Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan. Department of Biological Production, Akita Prefectural University, Akita, 010-0195, Japan. Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan. asano@pu-toyama.ac.jp. Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan. asano@pu-toyama.ac.jp" |
| DOI: | 10.1007/s11103-016-0459-6 |
| ISSN/ISBN: | 1573-5028 (Electronic) 0167-4412 (Linking) |
| Abstract: | "The giant knotweed Fallopia sachalinensis (Polygonaceae) synthesizes phenylacetonitrile (PAN) from L-phenylalanine when infested by the Japanese beetle Popillia japonica or treated with methyl jasmonate (MeJA). Here we identified (E/Z)-phenylacetaldoxime (PAOx) as the biosynthetic precursor of PAN and identified a cytochrome P450 that catalysed the conversion of (E/Z)-PAOx to PAN. Incorporation of deuterium-labelled (E/Z)-PAOx into PAN emitted from the leaves of F. sachalinensis was detected using gas chromatography-mass spectrometry. Further, using liquid chromatography-tandem mass spectrometry, we detected the accumulation of (E/Z)-PAOx in MeJA-treated leaves. These results showed that (E/Z)-PAOx is the biosynthetic precursor of PAN. MeJA-induced mRNAs were analysed by differential expression analysis using a next-generation sequencer. Of the 74,329 contigs obtained from RNA-seq and de novo assembly, 252 contigs were induced by MeJA treatment. Full-length cDNAs encoding MeJA-induced cytochrome P450s CYP71AT96, CYP82AN1, CYP82D125 and CYP715A35 were cloned using 5'- and 3'-RACE and were expressed using a baculovirus expression system. Among these cytochrome P450s, CYP71AT96 catalysed the conversion of (E/Z)-PAOx to PAN in the presence of NADPH and a cytochrome P450 reductase. It also acted on (E/Z)-4-hydroxyphenylacetaldoxime and (E/Z)-indole-3-acetaldoxime. The broad substrate specificity of CYP71AT96 was similar to that of aldoxime metabolizing cytochrome P450s. Quantitative RT-PCR analysis showed that CYP71AT96 expression was highly induced because of treatment with MeJA as well as feeding by the Japanese beetle. These results indicate that CYP71AT96 likely contributes the herbivore-induced PAN biosynthesis in F. sachalinensis" |
| Keywords: | Acetates/pharmacology Acetonitriles/*metabolism Animals Catalysis Coleoptera Cyclopentanes/pharmacology Cytochrome P-450 Enzyme System/genetics/*metabolism Fallopia/drug effects/*metabolism/physiology Gas Chromatography-Mass Spectrometry Gene Expression R; |
| Notes: | "MedlineYamaguchi, Takuya Noge, Koji Asano, Yasuhisa eng Netherlands 2016/03/02 Plant Mol Biol. 2016 Jun; 91(3):229-39. doi: 10.1007/s11103-016-0459-6. Epub 2016 Feb 29" |
