Preisig-Müller R, Gehlert R, Melchior F, Stietz U, Kindl H
Fachbereich Chemie, Philipps-Universität, D-35032 Marburg, Germany.
Biochemistry. 1997 Jul 8;36(27):8349-58. doi: 10.1021/bi970368h.
Stilbene synthases and the related bibenzyl synthases are plant polyketide synthases whose biological functions lie in the formation of antimicrobial phytoalexins. The formation of hydroxystilbenes from one molecule of acyl-CoA and three molecules of malonyl-CoA is catalyzed by a homodimeric 90 kDa protein and includes Claisen condensations and cleavage of a thioester followed by decarboxylation. Combining inhibitor studies, protein modifications, and site-directed mutagenesis, we were able to differentiate between the binding sites for malonyl-CoA and the regions responsible for the selection of the primer, p-coumaroyl-CoA or m-hydroxyphenylpropionyl-CoA, respectively. Mutations in the C-terminal part of the molecule or modification by photolabeling with p-azidocinnamoyl-CoA influence the overall reaction, the formation of hydroxystilbenes, but leave partial reactions, such as the malonyl-CoA:CO2 exchange and the malonyl-CoA-dependent modification of the enzyme, unaffected. Data obtained with several kinds of stilbene synthase and mutant forms suggest that the malonyl-CoA-dependent covalent modification takes place at a cysteine residue in the N-terminal part of the enzyme. Mutations in the C-terminal half of the enzyme molecule do not interfere with the malonyl-CoA-dependent reactions.
芪合酶及相关的联苄合酶是植物聚酮合酶，其生物学功能在于形成抗菌植物抗毒素。一分子酰基辅酶A和三分子丙二酰辅酶A形成羟基芪的过程由一种90 kDa的同二聚体蛋白催化，包括克莱森缩合反应、硫酯的裂解以及随后的脱羧反应。通过结合抑制剂研究、蛋白质修饰和定点诱变，我们能够区分丙二酰辅酶A的结合位点以及分别负责选择引物对香豆酰辅酶A或间羟基苯丙酰辅酶A的区域。分子C末端部分的突变或用对叠氮肉桂酰辅酶A进行光标记修饰会影响整体反应，即羟基芪的形成，但不会影响诸如丙二酰辅酶A与二氧化碳的交换以及丙二酰辅酶A依赖的酶修饰等部分反应。用几种芪合酶及其突变形式获得的数据表明，丙二酰辅酶A依赖的共价修饰发生在酶N末端部分的一个半胱氨酸残基处。酶分子C末端一半的突变不会干扰丙二酰辅酶A依赖的反应。
