Single point mutation abolishes water capture in germacradien‐4‐ol synthase

González Requena, Víctor, Srivastava, Prabhakar L. ORCID: https://orcid.org/0000-0002-8219-6419, Miller, David J. and Allemann, Rudolf K. ORCID: https://orcid.org/0000-0002-1323-8830 2024. Single point mutation abolishes water capture in germacradien‐4‐ol synthase. ChemBioChem , e202400290. 10.1002/cbic.202400290

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Abstract

The high‐fidelity sesquiterpene cyclase (−)‐germacradien‐4‐ol synthase (GdolS) converts farnesyl diphosphate into the macrocyclic alcohol (−)‐germacradien‐4‐ol. Site‐directed mutagenesis was used to decipher the role of key residues in the water control mechanism. Replacement of Ala176, located in the G1/2 helix, with non‐polar aliphatic residues of increasing size (valine, leucine, isoleucine and methionine) resulted in the accumulation of the non‐hydroxylated products germacrene A and germacrene D. In contrast, hydroxylation was maintained when the polar residues threonine, glutamine or aspartate replaced Ala176. Additionally, although a contribution of His150 to the nucleophilic water addition could be ruled out, the imidazole ring of His150 appears to assist carbocation stabilisation. The results presented here shed light on how hydroxylating sesquiterpene synthases can be engineered to design modified sesquiterpene synthases to reduce the need for further steps in the biocatalytic production of oxygenated sesquiterpenoids.

Item Type:	Article
Date Type:	Published Online
Status:	In Press
Schools:	Chemistry
Additional Information:	License information from Publisher: LICENSE 1: URL: http://creativecommons.org/licenses/by/4.0/
Publisher:	Wiley
ISSN:	1439-4227
Date of First Compliant Deposit:	8 August 2024
Date of Acceptance:	21 June 2024
Last Modified:	19 Sep 2024 12:59
URI:	https://orca.cardiff.ac.uk/id/eprint/171250

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