Peters R J, Ravn M M, Coates R M, Croteau R B
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
J Am Chem Soc. 2001 Sep 19;123(37):8974-8. doi: 10.1021/ja010670k.
Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinct cyclizations in the conversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial step of resin acid biosynthesis in grand fir (Abies grandis). The first reaction converts geranylgeranyl diphosphate to the stable bicyclic intermediate (+)-copalyl diphosphate via protonation-initiated cyclization. In the second reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone, and is directly coupled to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane family of diterpenes. Using the transition-state analogue inhibitor 14,15-dihydro-15-azageranylgeranyl diphosphate, it was demonstrated that each reaction of abietadiene synthase is carried out at a distinct active site. Mutations in two aspartate-rich motifs specifically delete one or the other activity and the location of these motifs suggests that the two active sites reside in separate domains. These mutants effectively complement each other, suggesting that the copalyl diphosphate intermediate diffuses between the two active sites in this monomeric enzyme. Free copalyl diphosphate was detected in steady-state kinetic reactions, thus conclusively demonstrating a free diffusion transfer mechanism. In addition, both mutant enzymes enhance the activity of wild-type abietadiene synthase with geranylgeranyl diphosphate as substrate. The implications of these results for the kinetic mechanism of abietadiene synthase are discussed.
枞酸型二烯合酶（AS）催化牻牛儿基牻牛儿基二磷酸转化为枞酸型二烯双键异构体混合物的过程中两个连续的、机制不同的环化反应，这是大冷杉（Abies grandis）树脂酸生物合成的第一步。第一个反应通过质子化引发的环化将牻牛儿基牻牛儿基二磷酸转化为稳定的双环中间体（+）-贝壳杉烯二磷酸。在第二个反应中，二磷酸酯离子化引发的环化产生三环全氢菲型骨架，并直接与一个1,2-甲基迁移偶联，该迁移产生了枞烷型二萜家族特有的C13异丙基。使用过渡态类似物抑制剂14,15-二氢-15-氮杂牻牛儿基牻牛儿基二磷酸，证明了枞酸型二烯合酶的每个反应都是在不同的活性位点进行的。两个富含天冬氨酸基序中的突变特异性地消除了其中一种或另一种活性，这些基序的位置表明两个活性位点位于不同的结构域。这些突变体有效地相互补充，表明贝壳杉烯二磷酸中间体在这种单体酶的两个活性位点之间扩散。在稳态动力学反应中检测到了游离的贝壳杉烯二磷酸，从而确凿地证明了一种自由扩散转移机制。此外，两种突变酶都增强了以牻牛儿基牻牛儿基二磷酸为底物的野生型枞酸型二烯合酶的活性。讨论了这些结果对枞酸型二烯合酶动力学机制的影响。
