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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
Author(s) -
Raiber Sigrid,
Schröder Gudrun,
Schröder Joachim
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00199-j
Subject(s) - enzyme , biochemistry , escherichia coli , chemistry , heterologous expression , mutagenesis , amino acid , complementation , biology , stereochemistry , gene , mutation , mutant , recombinant dna
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.