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Structural basis of catalysis in the bacterial monoterpene synthases linalool synthase and 1,8-cineole synthase

DOI: 10.1021/acscatal.7b01924 DOI Help

Authors: Vijaykumar Karuppiah (Manchester Institute of Biotechnology, University of Manchester) , Kara Ranaghan (University of Bristol) , Nicole G. H. Leferink (Manchester Institute of Biotechnology, University of Manchester) , Linus O. Johannissen (Manchester Institute of Biotechnology, University of Manchester) , Muralidharan Shanmugam (Manchester Institute of Biotechnology, University of Manchester) , Aisling Ni Cheallaigh (Manchester Institute of Biotechnology, University of Manchester) , Nathan Bennett (Manchester Institute of Biotechnology, University of Manchester) , Lewis Kearsey (Manchester Institute of Biotechnology, University of Manchester) , Eriko Takano (Manchester Institute of Biotechnology, University of Manchester) , John Gardiner (Manchester Institute of Biotechnology, University of Manchester) , Marc Van Der Kamp (University of Bristol) , Sam Hay (Manchester Institute of Biotechnology, University of Manchester) , Adrian J. Mulholland (University of Bristol) , David Leys (Manchester Institute of Biotechnology, University of Manchester) , Nigel S. Scrutton (Manchester Institute of Biotechnology, University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: August 2017
Diamond Proposal Number(s): 12788

Abstract: Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavours and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS) and show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterised sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes towards alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids.

Journal Keywords: monoterpene synthase; monoterpenoids; sesquiterpene synthase; terpenes; protein crystallography; molecular dynamics simulations

Subject Areas: Chemistry, Biology and Bio-materials


Instruments: I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography