Please use this identifier to cite or link to this item:
https://hdl.handle.net/2440/120062
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Type: | Journal article |
Title: | Selective hydroxylation of 1,8- and 1,4-cineole using bacterial P450 variants |
Author: | Lee, J.H.Z. Wong, S.H. Stok, J.E. Bagster, S.A. Beckett, J. Clegg, J.K. Brock, A.J. De Voss, J.J. Bell, S.G. |
Citation: | Archives of Biochemistry and Biophysics, 2019; 663:54-63 |
Publisher: | Elsevier |
Issue Date: | 2019 |
ISSN: | 0003-9861 1096-0384 |
Statement of Responsibility: | Joel H.Z. Lee, Siew Hoon Wong, Jeanette E. Stok, Sarah A. Bagster, James Beckett, Jack K. Clegg, Aidan J. Brock, James J. De Voss, Stephen G. Bell |
Abstract: | This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8- and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4- and 1,8-cineole. The CYP102A1 R47L/Y51F/A74G/F87V/L188Q mutant generated predominantly (1S)-6α-hydroxy-1,8-cineole (78% e.e.) from 1,8-cineole. Oxidation of 1,4-cineole by the CYP102A1 R47L/Y51F/F87A/I401P variant generated the 3α product in >90% yield. WT CYP101A1 formed a mixture metabolites with 1,8-cineole and very little product was generated with 1,4-cineole. In contrast the F87W/Y96F/L244A/V247L and F87W/Y96F/L244A variants of CYP101A1 favoured formation of 5α-hydroxy-1,8-cineole (>88%, 1S 86% e.e.) while the F87V/Y96F/L244A variant generated (1S)-6α-hydroxy-1,8-cineole in excess (90% regioselective, >99% e.e.). The CYP101A1 F87W/Y96F/L244A/V247L and F87W/Y96F/L244A mutants improved the oxidation of 1,4-cineole generating an excess of the 3α metabolite (1S > 99% e.e. with the latter). The CYP101A1 F87L/Y96F variant also improved the oxidation of this substrate but shifted the site of oxidation to the isopropyl group, (8-hydroxy-1,4-cineole). When this 8-hydroxy metabolite was generated in significant quantities desaturation of C8C9 to the corresponding alkene was also detected. |
Keywords: | Enzyme catalysis Cytochrome P450 Cineole Hydroxylation C-H bond oxidation |
Rights: | © 2018 Elsevier Inc. All rights reserved. |
DOI: | 10.1016/j.abb.2018.12.025 |
Grant ID: | http://purl.org/au-research/grants/arc/DP140103229 http://purl.org/au-research/grants/arc/FT140100355 |
Published version: | http://dx.doi.org/10.1016/j.abb.2018.12.025 |
Appears in Collections: | Aurora harvest 4 Chemistry publications |
Files in This Item:
File | Description | Size | Format | |
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hdl_120062.pdf | Accepted version | 1.16 MB | Adobe PDF | View/Open |
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