Abietadiene synthase catalysis: Mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement
Abietadiene synthase catalysis: Mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement
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DOI：
10.1073/pnas.022627099
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发表时间：
2002-01-22
影响因子：
11.1
通讯作者：
Croteau, RB
中科院分区：
文献类型：
--
作者：
Peters, RJ;Croteau, RB
冷杉合酶催化树脂酸生物合成中的关键步骤，通过两个顺序的、机械上不同的环化在不同的活性位点形成冷杉双键异构体的混合物。第一个反应，质子化引发的环化，将通用的二萜前体香叶基香叶基二磷酸转化为稳定的双环中间体柯巴基二磷酸。在第二，镁离子依赖性反应，二磷酸酯电离引发的环化产生的三环全氢菲型骨干，并耦合，通过分子内质子转移内的瞬态pimarenyl中间体，1，2-甲基迁移，产生的C13异丙基的松香烷结构的特征。替代脱质子化的终端松香烯基碳阳离子提供的混合物的松香，左旋海松二烯，和neoabieglycine，该产品的配置文件作为pH值的函数而变化。突变分析的氨基酸在活性位点的一个模型结构已确定的残基催化的关键，以及几个发挥作用，在指定产品的形成，显然是通过连接的镁离子辅因子。这些结果强烈表明，选择的替代品之间的去质子化的松香烯基中间体更多地取决于定位效果的碳阳离子-二磷酸阴离子反应伙伴比多个参与基地的pKa。在一个极端的情况下，突变体N765 A不能介导分子内质子转移，并中止反应，而不催化1，2-甲基迁移，仅产生sandaracopimaradiene，从而为反应途径的隐蔽pimarenyl中间体的相应立体化学提供支持证据。
Abietadiene synthase catalyzes the committed step in resin acid biosynthesis, forming a mixture of abietadiene double-bond isomers by two sequential, mechanistically distinct cyclizations at separate active sites. The first reaction, protonation-initiated cyclization, converts the universal diterpene precursor geranylgeranyl diphosphate to the stable bicyclic intermediate copalyl diphosphate. In the second, magnesium ion-dependent reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone and is coupled, by intramolecular proton transfer within a transient pimarenyl intermediate, to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane structure. Alternative deprotonations of the terminal abietenyl carbocation provide a mixture of abietadiene, levopimaradiene, and neoabietadiene, and this product profile varies as a function of pH. Mutational analysis of amino acids at the active site of a modeled structure has identified residues critical for catalysis, as well as several that play roles in specifying product formation, apparently by ligation of a magnesium ion cofactor. These results strongly suggest that choice between alternatives for deprotonation of the abietenyl intermediate depends more on the positioning effects of the carbocation–diphosphate anion reaction partners than on the pKa of multiple participating bases. In one extreme case, mutant N765A is unable to mediate the intramolecular proton transfer and aborts the reaction, without catalyzing 1,2-methyl migration, to produce only sandaracopimaradiene, thereby providing supporting evidence for the corresponding stereochemistry of the cryptic pimarenyl intermediate of the reaction pathway.