Molecular Cloning and Functional Characterization of Three Distinct N-Methyltransferases Involved in the Caffeine Biosynthetic Pathway in Coffee Plants
Molecular Cloning and Functional Characterization of Three Distinct N-Methyltransferases Involved in the Caffeine Biosynthetic Pathway in Coffee Plants
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咖啡植物咖啡因生物合成途径中三种不同 N-甲基转移酶的分子克隆和功能表征
DOI：
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
H. Sano
中科院分区：
文献类型：
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作者：
H. Uefuji;S. Ogita;Y. Yamaguchi;N. Koizumi;H. Sano
咖啡因是由黄苷通过 N-甲基化和核糖去除步骤合成的。在本研究中，从咖啡（Coffea arabica）植物的未成熟果实中分离出三种编码N-甲基转移酶的cDNA，并分别命名为CaXMT1、CaMXMT2和CaDXMT1。细菌表达的编码蛋白的催化特性被表征。 CaXMT1 催化黄嘌呤形成 7-甲基黄嘌呤，Km 值为 78 mm；CaMXMT2 催化 7-甲基黄嘌呤形成 3,7-二甲基黄嘌呤（可可碱），Km 为 251 mm；CaDXMT1 催化 Km 为 251 mm，催化形成 1,3,7-三甲基黄嘌呤（咖啡因）。 3,7-二甲基黄嘌呤，Km 为 1,222 mm。大肠杆菌的粗提物被发现可以催化去除 7-甲基黄嘌呤核糖部分，从而产生 7-甲基黄嘌呤。结果，当所有三种重组蛋白和大肠杆菌提取物混合时，黄苷在体外成功转化为咖啡因。 CaDXMT1 的转录本主要在未成熟的果实中积累，而 CaXMT1 和 CaMXMT2 的转录本则更广泛地在叶子、花芽和未成熟的果实等位点中检测到。这些结果表明，目前鉴定的三种N-甲基转移酶参与咖啡植物中的咖啡因生物合成，并证实了所提出的咖啡因生物合成途径：黄苷3 7-甲基黄苷3 7-甲基黄嘌呤3 可可碱3 咖啡因。
Caffeine is synthesized from xanthosine through N-methylation and ribose removal steps. In the present study, three types of cDNAs encoding N-methyltransferases were isolated from immature fruits of coffee (Coffea arabica) plants, and designated as CaXMT1, CaMXMT2, and CaDXMT1, respectively. The bacterially expressed encoded proteins were characterized for their catalytic properties. CaXMT1 catalyzed formation of 7-methylxanthosine from xanthosine with a Km value of 78 mm, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a Km of 251 mm, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with a Km of 1,222 mm. The crude extract of Escherichia coli was found to catalyze removal of the ribose moiety from 7-methylxanthosine, leading to the production of 7-methylxanthine. As a consequence, when all three recombinant proteins and E. coli extract were combined, xanthosine was successfully converted into caffeine in vitro. Transcripts for CaDXMT1 were predominantly found to accumulate in immature fruits, whereas those for CaXMT1 and CaMXMT2 were more broadly detected in sites encompassing the leaves, floral buds, and immature fruits. These results suggest that the presently identified three N-methyltransferases participate in caffeine biosynthesis in coffee plants and substantiate the proposed caffeine biosynthetic pathway: xanthosine3 7-methylxanthosine 3 7-methylxanthine 3 theobromine 3 caffeine.
影响因子：
7.4
作者：
Ashihara, H;Monteiro, AM;Crozier, A
通讯作者：
Crozier, A