Ergot alkaloid biosynthesis in Aspergillus fumigatus -: Overproduction and biochemical characterization of a 4-dimethylallyltryptophan N-methyltransferase
Ergot alkaloid biosynthesis in Aspergillus fumigatus -: Overproduction and biochemical characterization of a 4-dimethylallyltryptophan N-methyltransferase
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DOI：
10.1074/jbc.m804979200
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发表时间：
2008-10-03
影响因子：
4.8
通讯作者：
Li, Shu-Ming
中科院分区：
文献类型：
--
作者：
Rigbers, Ole;Li, Shu-Ming
推定基因fgaMT在烟曲霉烟曲霉生物合成基因簇中被鉴定。通过 PCR 从 cDNA 文库中扩增 fgaMT 的编码区，克隆到 pQE60 中，并在大肠杆菌中过表达。 FgaMT包含339个氨基酸，分子量约为38.1 kDa。将可溶性二聚体 His(6)-FgaMT 纯化至接近均质并进行生化表征。发现 FgaMT 在 S-腺苷甲硫氨酸存在下催化 4-二甲基烯丙基色氨酸的 N-甲基化，导致形成 4-二甲基烯丙基-L-abrine，并通过 NMR 和质谱分析进行鉴定。因此，FgaMT 代表麦角生物碱生物合成中的第二种途径特异性酶。该酶的酶促反应不需要金属离子，并且对吲哚环 C-4 位异戊二烯基部分表现出相对较高的特异性。吲哚环修饰的 4-二甲基烯丙基色氨酸衍生物也被 FgaMT 接受作为底物。 4-二甲基烯丙基色氨酸和 S-腺苷甲硫氨酸的 Km 值分别测定为 0.12 和 2.4 mM。周转次数为2.0 s(-1)。
The putative gene fgaMT was identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus. The coding region of fgaMT was amplified by PCR from a cDNA library, cloned into pQE60, and overexpressed in Escherichia coli. FgaMT comprises 339 amino acids with a molecular mass of about 38.1 kDa. The soluble dimeric His(6)-FgaMT was purified to near homogeneity and characterized biochemically. FgaMT was found to catalyze the N-methylation of 4-dimethylallyltryptophan in the presence of S-adenosylmethionine, resulting in the formation of 4-dimethylallyl-L-abrine, which was identified by NMR and mass spectrometry analysis. Therefore, FgaMT represents the second pathway-specific enzyme in the biosynthesis of ergot alkaloids. The enzyme did not require metal ions for its enzymatic reaction and showed a relatively high specificity toward the prenyl moiety at position C-4 of the indole ring. 4-Dimethylallyltryptophan derivatives with modification at the indole ring were also accepted by FgaMT as substrates. Km values for 4-dimethylallyltryptophan and S-adenosylmethionine were determined at 0.12 and 2.4 mM, respectively. The turnover number was 2.0 s(-1).