Caffeic acid O-methyltransferase is involved in the synthesis of melatonin by methylating N-acetylserotonin in Arabidopsis
Caffeic acid O-methyltransferase is involved in the synthesis of melatonin by methylating N-acetylserotonin in Arabidopsis
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DOI：
10.1111/jpi.12160
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发表时间：
2014-09-01
影响因子：
10.3
通讯作者：
Back, Kyoungwhan
中科院分区：
文献类型：
--
作者：
Byeon, Yeong;Lee, Hyoung Yool;Back, Kyoungwhan
尽管植物 N-乙酰血清素甲基转移酶 (ASMT) 最近从水稻中克隆出来，但双子叶植物中似乎不存在同源基因。为了从双子叶植物中从头克隆 ASMT，我们在大肠杆菌中表达了 8 个拟南芥 O-甲基转移酶 (OMT) cDNA，并通过测量应用 1 mM N-乙酰血清素 (NAS) 后褪黑激素的产生来筛选 ASMT 活性。在含有全长 cDNA 的八个菌株中，OMT3 菌株产生高水平的褪黑激素，表明 OMT3 编码活性 ASMT。 OMT3 已被称为咖啡酸 OMT (COMT)，表明该酶具有多种功能。纯化的重组拟南芥 COMT (AtCOMT) 显示出高 ASMT 活性，催化 NAS 转化为褪黑激素。 ASMT活性的K-m和V-max值分别为233μM和1800pmol/min/mg蛋白质，而COMT活性的K-m和V-max值分别为103μM和564,000pmol/min/mg蛋白质。 ASMT 活性的催化效率 (V-max/K-m) 比 COMT 低 709 倍。在体外，添加咖啡酸以剂量依赖性方式显着降低 ASMT 活性，但 NAS 不会改变 COMT 活性。最后，当用 1 mM NAS 浸润拟南芥叶子时，拟南芥 comt 敲除突变体表现出比野生型更少的褪黑激素产量，表明 COMT 在植物褪黑激素生物合成中的体内作用。
Although a plant N-acetylserotonin methyltransferase (ASMT) was recently cloned from rice, homologous genes appear to be absent in dicotyledonous plants. To clone an ASMT de novo from a dicotyledonous plant, we expressed eight Arabidopsis thaliana O-methyltransferase (OMT) cDNAs in Escherichia coli and screened for ASMT activity by measuring melatonin production after the application of 1 mM N-acetylserotonin (NAS). Among the eight strains harboring the full-length cDNAs, the OMT3 strain produced high levels of melatonin, suggesting that OMT3 encodes an active ASMT. OMT3 is already known as caffeic acid OMT (COMT), suggesting multiple functions for this enzyme. The purified recombinant A. thaliana COMT (AtCOMT) showed high ASMT activity, catalyzing the conversion of NAS to melatonin. The K-m and V-max values for ASMT activity were 233 mu M and 1800 pmol/min/mg protein, while the K-m and V-max values for COMT activity were 103 mu M and 564,000 pmol/min/mg protein, respectively. The catalytic efficiency (V-max/K-m) for ASMT activity was 709-fold lower than for COMT. In vitro, ASMT activity was dramatically decreased by the addition of caffeic acid in a dose-dependent manner, but the activity of COMT was not altered by NAS. Lastly, the Arabidopsis comt knockout mutant exhibited less production of melatonin than the wild type when Arabidopsis leaves were infiltrated with 1 mM NAS, suggestive of in vivo role of COMT in melatonin biosynthesis in plants.