Single Point Mutation Abolishes Water Capture in Germacradien-4-ol Synthase

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DOI

  • Víctor González Requena
    Cardiff University
  • Prabhakar L Srivastava
    Cardiff University
  • David J Miller
    Cardiff University
  • Rudolf K Allemann
    Cardiff University

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.

Keywords

  • Water/chemistry, Point Mutation, Mutagenesis, Site-Directed, Sesquiterpenes, Germacrane/metabolism
Original languageEnglish
Pages (from-to)e202400290
JournalChemBioChem
Volume25
Issue number23
Early online date7 Aug 2024
DOIs
Publication statusPublished - 2 Dec 2024
Externally publishedYes
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