The Catalytic Potential of Coptis japonica NCS2 Revealed - Development and Utilisation of a Fluorescamine-Based Assay

被引：65

作者：

Pesnot, Thomas ^{[1
]}

Gershater, Markus C. ^{[2
]}

Ward, John M. ^{[2
]}

Hailes, Helen C. ^{[1
]}

机构：

[1] UCL, Dept Chem, Christopher Ingold Labs, London WC1H 0AJ, England

[2] UCL, Res Dept Struct & Mol Biol, ISMB, London WC1E 6BT, England

来源：

ADVANCED SYNTHESIS & CATALYSIS | 2012年 / 354卷 / 16期

基金：

英国生物技术与生命科学研究理事会;

关键词：

alkaloids; biotransformations; norcoclaurine synthase; Pictet-Spengler reaction; tetrahydroisoquinolines; BENZYLISOQUINOLINE ALKALOID BIOSYNTHESIS; 1ST COMMITTED STEP; NORCOCLAURINE SYNTHASE; STRICTOSIDINE SYNTHASE; PRIMARY AMINES; ENZYME; PURIFICATION; ISOQUINOLINE; EXPRESSION; MECHANISM;

D O I：

10.1002/adsc.201200641

中图分类号：

O69 [应用化学];

学科分类号：

081704 ;

摘要：

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.

引用

页码：2997 / 3008

页数：12

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