title: Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function.
abstract: Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.
authors.affiliation: Department of Natural Products and Biotechnology, Mediterranean Agronomic Institute of Chania, Chania, Greece. sotirios@maich.gr
authors.forename: Sotirios C
authors.initials: SC
authors.lastname: Kampranis
authors.forename: Daphne
authors.initials: D
authors.lastname: Ioannidis
authors.forename: Alan
authors.initials: A
authors.lastname: Purvis
authors.forename: Walid
authors.initials: W
authors.lastname: Mahrez
authors.forename: Ederina
authors.initials: E
authors.lastname: Ninga
authors.forename: Nikolaos A
authors.initials: NA
authors.lastname: Katerelos
authors.forename: Samir
authors.initials: S
authors.lastname: Anssour
authors.forename: Jim M
authors.initials: JM
authors.lastname: Dunwell
authors.forename: Jörg
authors.initials: J
authors.lastname: Degenhardt
authors.forename: Antonios M
authors.initials: AM
authors.lastname: Makris
authors.forename: Peter W
authors.initials: PW
authors.lastname: Goodenough
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Johnson
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002849:Chromatography, Gas; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D027544:Salvia / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014835:Volatilization
chemical_list: D019883:Alkyl and Aryl Transferases; C108877:terpene synthase