Cloning and functional characterization of the Arabidopsis N-acetylserotonin O-methyltransferase responsible for melatonin synthesis
Cloning and functional characterization of the Arabidopsis N-acetylserotonin O-methyltransferase responsible for melatonin synthesis
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DOI：
10.1111/jpi.12289
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发表时间：
2016-01-01
影响因子：
10.3
通讯作者：
Back, Kyoungwhan
中科院分区：
文献类型：
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作者：
Byeon, Yeong;Lee, Hye-Jung;Back, Kyoungwhan
n -乙酰5 -羟色胺o -甲基转移酶（ASMT）基因编码催化n -乙酰5 -羟色胺转化为褪黑激素的酶，这是褪黑激素生物合成的最后一步。第一个克隆的植物ASMT基因来自水稻。最近从苹果（Malus zumi）中分离到一个与水稻ASMT蛋白同源性仅为39.7%的同源基因。苹果ASMT序列的低同源性促使我们在拟南芥基因组中筛选同源ASMT基因。与水稻ASMT基因序列同源性最高的是At4g35160基因（31%），其次是At1g76790基因（29%）。我们纯化了两个拟南芥基因表达的重组蛋白。重组蛋白At4g35160具有ASMT酶活性，而重组蛋白At1g76790不具有ASMT酶活性；因此，我们将At4g35160定位为拟南芥ASMT （AtASMT）基因。AtASMT蛋白催化n -乙酰5-羟色胺转化为褪黑素和5-甲氧基色胺，其V-max值分别为0.11和0.29 pkat/mg蛋白。然而，AtASMT没有咖啡酸o -甲基转移酶活性，表明其功能对褪黑激素合成具有高度特异性。镉处理诱导拟南芥AtASMT转录本，随后增加褪黑激素合成。与其他ASMT蛋白类似，AtASMT定位于细胞质中，其在水稻中的异位过表达导致ASMT酶活性增加和褪黑激素的产生，表明AtASMT参与褪黑激素的合成。
The N-acetylserotonin O-methyltransferase (ASMT) gene encodes the enzyme that catalyzes the conversion of N-acetylserotonin to melatonin as the last step in melatonin biosynthesis. The first plant ASMT gene to be cloned was from rice. An orthologous gene encoding a protein with ASMT activity and only 39.7% amino acid sequence identity to the rice ASMT protein was recently isolated from apple (Malus zumi). The low homology of the apple ASMT sequence prompted us to screen the Arabidopsis genome for a homologous ASMT gene. The At4g35160 gene exhibited the highest sequence identity (31%) to the rice ASMT gene, followed by the At1g76790 gene with 29% sequence identity. We purified recombinant proteins expressed from the two Arabidopsis genes. The At4g35160 recombinant protein exhibited ASMT enzyme activity, but the At1g76790 recombinant protein did not; thus, we designated At4g35160 as an Arabidopsis thaliana ASMT (AtASMT) gene. The AtASMT protein catalyzed the conversion of N-acetylserotonin to melatonin and serotonin to 5-methoxytryptamine with V-max values of 0.11 and 0.29 pkat/mg protein, respectively. However, AtASMT exhibited no caffeic acid O-methyltransferase activity, suggesting that its function was highly specific to melatonin synthesis. AtASMT transcripts were induced by cadmium treatment in Arabidopsis followed by increased melatonin synthesis. Similar to other ASMT proteins, AtASMT was localized in the cytoplasm and its ectopic overexpression in rice resulted in increased ASMT enzyme activity and melatonin production, indicating the involvement of AtASMT in melatonin synthesis.