title: Molecular and enzymatic characterization of two stilbene synthases from Eastern white pine (Pinus strobus). A single Arg/His difference determines the activity and the pH dependence of the enzymes.
abstract: Pinus strobus (Eastern white pine) contains stilbenes biosynthetically derived from cinnamoyl-CoA (pinosylvin) or dihydrocinnamoyl-CoA (dihydropinosylvin). We screened a P. strobus cDNA library with a stilbene synthase (STS) probe from Pinus sylvestris. The eight isolated cDNAs represented two closely related STS genes with five amino acid differences in the proteins. The enzyme properties were investigated after heterologous expression in Escherichia coli. Both proteins preferred cinnamoyl-CoA against dihydrocinnamoyl-CoA and thus represented pinosylvin synthases. Otherwise they revealed large differences. STS1 had only 3-5% of the activity of STS2, its pH optimum was shifted to lower values (pH 6), and it synthesized with cinnamoyl-CoA a second unknown product. Site-directed mutagenesis demonstrated that a single Arg-to-His exchange in STS1 was responsible for all of the differences. The proton acceptor properties of His are discussed as the reason for the properties of STS1.
authors.affiliation: Institut für Biologie II, Universität Freiburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Raiber
authors.forename: G
authors.initials: G
authors.lastname: Schröder
authors.forename: J
authors.initials: J
authors.lastname: Schröder
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014197:Trees / Q000201:enzymology*
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; D000217:Acyltransferases; C022674:stilbene synthase