The Catalytic Potential of Coptis japonica NCS2 Revealed - Development and Utilisation of a Fluorescamine-Based Assay
The Catalytic Potential of Coptis japonica NCS2 Revealed - Development and Utilisation of a Fluorescamine-Based Assay
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DOI：
10.1002/adsc.201200641
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发表时间：
2012-11-01
影响因子：
5.4
通讯作者：
Hailes, Helen C.
中科院分区：
文献类型：
--
作者：
Pesnot, Thomas;Gershater, Markus C.;Hailes, Helen C.
研究人员对来自黄连 NCS2 的去甲乌云碱合酶 (NCS) 的多功能性和潜力进行了研究，并开发和应用了一种使用近四十种胺/醛的新型荧光高通量测定方法。 CjNCS2 对选定的非天然底物所施加的立体控制已被确定，其中四氢异喹啉 (THIA) 形成为 (1S)-异构体，其含量 > 95% ee，正如天然产物去甲乌云碱所观察到的那样。利用已报道的唐松草NCS X射线晶体结构，进行涉及THIA机制中间体的对接计算，并结合CjNCS2筛选结果，进一步了解NCS的作用模式。这些发现表明，除了关键活性位点残基 K122 和 E110 之外，D141 在机制上对于酶促转化也至关重要。我们提出的醛从酶口袋中突出的机制进一步支持了 NCS 对醛底物的特殊耐受性。
The versatility and potential of a norcoclaurine synthase (NCS) from Coptis japonica NCS2 has been investigated, together with the development and application of a novel fluorescence-based high-throughput assay using nearly forty amines/aldehydes. The stereocontrol exerted by CjNCS2 on selected non-natural substrates has been determined, where the tetrahydroisoquinolines (THIAs) were formed as the (1S)-isomer in >95% ee, as observed with the natural product norcoclaurine. Docking calculations involving THIA mechanism intermediates, utilising the reported Thalictrum flavum NCS X-ray crystallographic structure, were carried out and combined with the CjNCS2 screening results to further understand the mode of action of NCS. These findings suggested that in addition to the key active-site residues K122 and E110, D141 is also mechanistically essential for the enzymatic transformation. The exceptional tolerance of NCS towards aldehyde substrates is furthermore supported by our proposed mechanism in which the aldehydes protrude out of the enzymatic pocket.