title: Two residues determine the product profile of the class II diterpene synthases TPS14 and TPS21 of Tripterygium wilfordii.
abstract: The medicinal plant Tripterygium wilfordii (Celastraceae) contains a pair of class II diterpene synthases (diTPS) of specialized labdane-type metabolism that, despite remarkably close homology, form strikingly different products. TwTPS21 catalyzes bicyclization of the linear C20 precursor geranylgeranyl diphosphate to ent-copal-8-ol diphosphate, while TwTPS14 forms kolavenyl diphosphate. To determine the amino acid signature controlling the functional divergence of the homologues, we modeled their structures based on an existing crystal structure of the Arabidopsis ent-copalyl diphosphate synthase, archetypal of diTPSs in general metabolism of gibberellin phytohormones. Of the residues differing between TwTPS21 and TwTPS14 two located to the predicted active site, and we hypothesized that these are responsible for the functional differentiation of the enzymes. Using site-directed mutagenesis, we generated a panel of six variants, where one, or both positions were exchanged between the enzymes. In coupled heterologous assays with a corresponding class I diTPS, TwTPS2, complete product interchange was observed in variants with both reciprocal mutations, while substitutions of either residue gave mixed product profiles. Two mutants, TwTPS14:Y265H and TwTPS21:A325V, also produced ent-copalyl diphosphate, highlighting the evolutionary potential of enzymes of this family to drive rapid diversification of plant diterpene biosynthesis through neo-functionalization. Our study contributes to the understanding of structure-function relation in plant class II diTPSs and complements previous mutational studies of Arabidopsis ent-copalyl diphosphate synthase with additional examples from the specialized metabolism of T. wilfordii.
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Denmark; Center for Synthetic Biology "bioSYNergy" and Copenhagen Plant Sciences Centre, Denmark.
authors.forename: Nikolaj L
authors.initials: NL
authors.lastname: Hansen
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Denmark; Center for Synthetic Biology "bioSYNergy" and Copenhagen Plant Sciences Centre, Denmark.
authors.forename: Jakob N
authors.initials: JN
authors.lastname: Nissen
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Denmark; Center for Synthetic Biology "bioSYNergy" and Copenhagen Plant Sciences Centre, Denmark. Electronic address: hamberge@msu.edu.
authors.forename: Björn
authors.initials: B
authors.lastname: Hamberger
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry*; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D029757:Tripterygium / Q000201:enzymology*
chemical_list: D010940:Plant Proteins; D019883:Alkyl and Aryl Transferases
keywords: Celastraceae; Diterpene synthase; Homology model; Structure-function relation; Tripterygium wilfordii