Bifunctional abietadiene synthase: Free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites
Bifunctional abietadiene synthase: Free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites
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DOI：
10.1021/ja010670k
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发表时间：
2001-09-19
影响因子：
15
通讯作者：
Croteau, RB
中科院分区：
文献类型：
--
作者：
Peters, RJ;Ravn, MM;Croteau, RB
Abietadiene synthase(AS)催化两个连续的、机械上不同的环化反应，将香叶基香叶基二磷酸转化为松二烯双键异构体的混合物，作为大冷杉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反应，将香叶基香叶基二磷酸转化为稳定的双环中间体(+)-二磷酸钴。在第二个反应中，二磷酸酯电离引发的环化反应生成三环全氢菲主链，并直接偶联到1，2-甲基迁移，生成松烷二萜家族特有的C13异丙基。使用过渡态类似物14，15-二氢-15-氮杂基香叶基二磷酸，证明了松二烯合成酶的每个反应都在不同的活性部位进行。两个富含天冬氨酸的基序的突变专门删除了一个或另一个活性，这些基序的位置表明两个活性位点位于不同的结构域中。这些突变体有效地相互补充，表明二磷酸铜酰中间体在该单体酶的两个活性部位之间扩散。在稳态动力学反应中检测到了游离的二磷酸钴，从而最终证明了自由扩散转移机理。此外，两种突变酶均能提高以香叶基香叶基二磷酸为底物的野生型松二烯合成酶的活性。讨论了这些结果对松二烯合成酶动力学机制的影响。
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.