Redesigning the molecular choreography to prevent hydroxylation in germacradien-11-ol synthase catalysis

et al.

Research output: Contribution to journalArticle (Academic Journal)peer-review

5 Citations (Scopus)
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Abstract

Natural sesquiterpene synthases have evolved to make complex terpenoids by quenching reactive carbocations either by proton transfer or by hydroxylation (water capture), depending on their active site. Germacradien-11-ol synthase (Gd11olS) from Streptomyces coelicolor catalyzes the cyclization of farnesyl diphosphate (FDP) into the hydroxylated sesquiterpene germacradien-11-ol. Here, we combine experiment and simulation to guide the redesign of its active site pocket to avoid hydroxylation of the product. Molecular dynamics simulations indicate two regions between which water molecules can flow that are responsible for hydroxylation. Point mutations of selected residues result in variants that predominantly form a complex nonhydroxylated product, which we identify as isolepidozene. Our results indicate how these mutations subtly change the molecular choreography in the Gd11olS active site and thereby pave the way for the engineering of terpene synthases to make complex terpenoid products.
Original languageEnglish
Pages (from-to)1033–1041
Number of pages9
JournalACS Catalysis
Volume11
Issue number3
DOIs
Publication statusPublished - 7 Jan 2021

Bibliographical note

Funding Information:
This work was supported by the BBSRC (grant numbers BB/R001596/1 and BB/R001332/1). M.W.v.d.K. is a BBSRC David Phillips Fellow (grant BB/M026280/1). Simulations were performed using the computational facilities of the Advanced Computing Research Centre, University of Bristol. We thank Dr. Robert Jenkins (Cardiff University) for his help with NMR spectroscopy. We also thank to Prof. John Pickett CBE, DSc, FRS (Cardiff University) for a critical reading of the manuscript and valuable suggestions.

Publisher Copyright:
© 2021 American Chemical Society. All rights reserved.

Keywords

  • terpene synthase
  • terpenoid
  • molecular dynamics
  • mutagenesis
  • enzyme redesign
  • enzyme mechanism

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