Raiber S, Schröder G, Schröder J
Institut für Biologie II, Universität Freiburg, Germany.
FEBS Lett. 1995 Mar 20;361(2-3):299-302. doi: 10.1016/0014-5793(95)00199-j.
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.
北美乔松（东部白松）含有通过生物合成从肉桂酰辅酶A（松柏醇）或二氢肉桂酰辅酶A（二氢松柏醇）衍生而来的芪类化合物。我们用来自欧洲赤松的芪合酶（STS）探针筛选了北美乔松的cDNA文库。分离出的八个cDNA代表了两个密切相关的STS基因，其蛋白质中有五个氨基酸差异。在大肠杆菌中进行异源表达后研究了酶的特性。两种蛋白质都更倾向于肉桂酰辅酶A而非二氢肉桂酰辅酶A，因此代表松柏醇合酶。否则它们显示出很大差异。STS1的活性仅为STS2的3 - 5%，其最适pH值向较低值（pH 6）偏移，并且它与肉桂酰辅酶A合成了另一种未知产物。定点诱变表明，STS1中单个精氨酸到组氨酸的交换是造成所有这些差异的原因。讨论了组氨酸的质子受体特性是STS1特性的原因。
