PDF(Pubmed)
Abstract：
Tryptophan decarboxylases (TDCs) are a group of pyridoxal 5\'-phosphate-dependent enzymes involved in the enzymatic conversion of tryptophan into tryptamine, a critical biogenic amine. We herein mined and cloned a TDC-encoding gene, CaTDC3, from camptothecin-producing plant Camptotheca acuminata. The intact CaTDC3 was heterologously overexpressed in Escherichia coli and the recombinant CaTDC3 was purified to homogeneity. High-performance liquid chromatography (HPLC)-diode array detector (DAD) and high resolution mass spectrometry (HRMS) data analyses of the CaTDC3-catalyzed reaction mixture confirmed the catalytically decarboxylative activity of CaTDC3. CaTDC3 shows strict stereoselectivity for L-tryptophan. Homology modeling and molecular docking implied CaTDC3\'s recognition of L-tryptophan derivatives and analogs. Substrate scope investigations revealed that the appropriate substituent groups on the indole ring, i.e., hydroxylated and halogenated L-tryptophans, could be recognized by CaTDC3 and the decarboxylation reactions generated the corresponding tryptamines. The Cβ -methyl-L-tryptophans were decarboxylated by CaTDC3 efficiently. 1-Thio-L-tryptophan, the NH group of the indole ring replaced by an S atom, could be decarboxylated by CaTDC3. CaTDC3 catalyzed the decarboxylation of 7-aza-L-tryptophan, an N displacement of the C on the aromatic ring, to afford 7-aza-tryptamine. L-Kynurenine, an L-tryptophan degradation product, could be decarboxylated by CaTDC3. The present works uncover a catalytically promiscuous TDC and the TDC is a versatile decarboxylase in synthetic biology for specialized pharmaceutically important substances.
摘要：
色氨酸脱羧酶（TDC）是一组吡哆醛5'-磷酸依赖性酶，参与色氨酸向色胺的酶转化，一种关键的生物胺.我们在此挖掘并克隆了TDC编码基因，CaTDC3，来自喜树碱生产植物喜树。完整的CaTDC3在大肠杆菌中异源过表达，重组CaTDC3纯化至均一。CaTDC3催化的反应混合物的高效液相色谱（HPLC）-二极管阵列检测器（DAD）和高分辨率质谱（HRMS）数据分析证实了CaTDC3的催化脱羧活性。CaTDC3显示对L-色氨酸的严格立体选择性。同源建模和分子对接暗示了CaTDC3对L-色氨酸衍生物和类似物的识别。底物范围研究表明，吲哚环上的适当取代基，即，羟基化和卤化L-色氨酸，可以被CaTDC3识别，脱羧反应产生相应的色胺。用CaTDC3有效地将Cβ-甲基-L-色氨酸脱羧。1-硫代-L-色氨酸,吲哚环的NH基团被S原子取代，可以被CaTDC3脱羧。CaTDC3催化7-氮杂-L-色氨酸脱羧,芳香环上C的N置换，提供7-氮杂-色胺.L-犬尿氨酸,一种L-色氨酸降解产物,可以被CaTDC3脱羧。本工作揭示了催化混杂的TDC，并且TDC是合成生物学中用于特殊药学上重要物质的通用脱羧酶。
