title: Characterization of the first naturally thermostable terpene synthases and development of strategies to improve thermostability in this family of enzymes.
abstract: The terpenoid family of natural products is being targeted for heterologous microbial production as a cheaper and more reliable alternative to extraction from plants. The key enzyme responsible for diversification of terpene structure is the class-I terpene synthase (TS), and these often require engineering to improve properties such as thermostability, robustness and catalytic activity before they are suitable for industrial use. Improving thermostability typically relies on screening a large number of mutants, as there are no naturally thermostable TSs described upon which to base rational design decisions. We have characterized the first examples of natural TSs exhibiting thermostability, which catalyse the formation of the sesquiterpene τ-muurolol at temperatures up to 78 °C. We also report an enzyme with a kcatvalue of 0.95 s-1at 65 °C, the highest kcatrecorded for a bacterial sesquiterpene synthase. In turn, these thermostable enzymes were used as a model to inform the rational engineering of another TS, with the same specificity but low sequence identity to the model. The newly engineered variant displayed increased thermostability and turnover. Given the high structural homology of the class-I TS domain, this approach could be generally applicable to improving the properties of other enzymes in this class.
  
  
  
  Model data are available in the PMDB database under the accession number PM0080780.
authors.affiliation: Department of Biology and Biochemistry, University of Bath, UK.
authors.forename: Matthew Q
authors.identifier: 0000-0002-5737-1174
authors.initials: MQ
authors.lastname: Styles
authors.affiliation: Department of Biology and Biochemistry, University of Bath, UK.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Nesbitt
authors.affiliation: Department of Biology and Biochemistry, University of Bath, UK.
authors.forename: Scott
authors.initials: S
authors.lastname: Marr
authors.affiliation: Department of Chemistry, University of Bath, UK.
authors.forename: Marc
authors.initials: M
authors.lastname: Hutchby
authors.affiliation: Department of Biology and Biochemistry, University of Bath, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Leak
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry*; D041862:Chloroflexi / Q000201:enzymology*; D004926:Escherichia coli; D006358:Hot Temperature; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000378:metabolism; D012717:Sesquiterpenes / Q000378:metabolism; D013329:Structure-Activity Relationship; D013729:Terpenes / Q000378:metabolism
chemical_list: D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D013729:Terpenes; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase
keywords: kinetics; structural model; terpenoid; thermophile; thermostability