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The Catalytic Potential of Coptis japonica NCS2 Revealed – Development and Utilisation of a Fluorescamine‐Based Assay
Author(s) -
Pesnot Thomas,
Gershater Markus C.,
Ward John M.,
Hailes Helen C.
Publication year - 2012
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201200641
Subject(s) - chemistry , aldehyde , stereochemistry , natural product , active site , docking (animal) , japonica , enzyme , catalysis , combinatorial chemistry , biochemistry , botany , medicine , nursing , biology
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 Cj NCS2 on selected non‐natural substrates has been determined, where the tetrahydroisoquinolines (THIAs) were formed as the (1 S )‐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 Cj NCS2 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.