Biosynthesis of monoterpenes: preliminary characterization of i-endo-fenchol synthetase from fennel (Foeniculum vulgare) and evidence that no free intermediate is involved in the cyclization of geranyl pyrophosphate to the rearranged product.
Biosynthesis of monoterpenes: preliminary characterization of i-endo-fenchol synthetase from fennel (Foeniculum vulgare) and evidence that no free intermediate is involved in the cyclization of geranyl pyrophosphate to the rearranged product.
复制标题
单萜的生物合成：来自茴香（Foeniculum vulgare）的 i-endo-fenchol 合成酶的初步表征，以及证据表明香叶基焦磷酸环化为重排产物时没有游离中间体参与。
DOI：
10.1016/0003-9861(80)90385-9
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发表时间：
1980
影响因子：
3.9
通讯作者：
R. Ronald
中科院分区：
文献类型：
--
作者：
R. Croteau;M. Felton;R. Ronald
茴香叶可溶性酶制剂已显示催化焦磷酸香叶酯和焦磷酸橙花酯二者的阳离子依赖性环化成双环重排的单萜-内-小茴香酚（R.克罗托湾Felton和R.罗纳德，1980年。200，524-533）。为了检查可能存在的游离中间体之间的无环前体和小茴香酚，并除去竞争的环化酶和焦磷酸酶的活动，可溶性制剂部分纯化硫酸铵分级，然后凝胶过滤Sephadex G-150和离子交换色谱上O-二乙基氨基乙基纤维素。香叶基焦磷酸和橙花基焦磷酸到小茴香酚的环化活性在色谱分离上是一致的，表明相同的酶能够环化两种无环底物。未检测到无环前体的相互转化。虽然焦磷酸双酯是一个自由的中间体，在生物合成的相关双环单萜醇β，蛋白质分馏和同位素稀释实验都排除outendo-fenchyl焦磷酸作为一个自由的中间体，在fenchol生物合成。类似地，虽然证实了茴香烷骨架的构建涉及中间体蒎烷骨架的重排，但同位素稀释实验排除了α-蒎烯、β-蒎烯、顺式-2-蒎醇、反式-2-蒎醇和相应的2-蒎基焦磷酸的光学对映体作为酶催化反应的游离中间体。此外，酶促反应产物的详尽搜索没有提供任何证据表明参与任何游离中间体之间的无环前体和小茴香酚。内切-小茴香酚合成酶具有60，000的表观分子量，显示接近7.0的最适pH，并且需要Mn 2+（1 mm）用于催化活性。Co ~（2+）可以部分取代Mn ~（2+），但其它二价阳离子则无效。部分纯化的合成酶被抑制byph-hydroxymercuribenzoate和苯乙二醛，它表现出优先选择香叶焦磷酸橙花焦磷酸作为底物。提出了一个小茴香酚合成酶催化环化和重排的集成方案。
A soluble enzyme preparation from the leaves of fennel (Foeniculum vulgareM.) has been shown to catalyze the cation-dependent cyclization of both geranyl pyrophosphate and neryl pyrophosphate to the bicyclic rearranged monoterpenel-endo-fenchol (R. Croteau, M. Felton, and R. Ronald, 1980Arch. Biochem. Biophys.200, 524–533). To examine the possible presence of free intermediates between the acyclic precursors and fenchol, and to remove competing cyclase and pyrophosphatase activities, the soluble preparation was partially purified by ammonium sulfate fractionation followed by gel filtration on Sephadex G-150 and ion exchange chromatography onO-diethylaminoethyl-cellulose. Activities for the cyclization of geranyl pyrophosphate and neryl pyrophosphate to fenchol were coincident on Chromatographic fractionation suggesting that the same enzyme was capable of cyclizing both acyclic substrates. No interconversion of the acyclic precursors was detected. Although bornyl pyrophosphate is a free intermediate in the biosynthesis of the related bicyclic monoterpenol borneol, both protein fractionation and isotopic dilution experiments ruled outendo-fenchyl pyrophosphate as a free intermediate in fenchol biosynthesis. Similarly, while construction of the fenchane skeleton was demonstrated to involve the rearrangement of an intermediate pinane skeleton, isotopic dilution experiments ruled out both optical antipodes of α-pinene, β-pinene,cis-2-pinanol,trans-2-pinanol, and the corresponding 2-pinyl pyrophosphates as free intermediates of the enzyme-catalyzed reaction. Furthermore, exhaustive search of the enzymatic reaction products provided no evidence to suggest the involvement of any free intermediate between the acyclic precursor and fenchol. Theendo-fenchol synthetase has an apparent molecular weight of 60,000, shows a pH optimum near 7.0, and requires Mn2+(1 mm) for catalytic activity. Co2+can partially substitute for Mn2+, but other divalent cations are ineffective. The partially purified synthetase is inhibited byp-hydroxymercuribenzoate and by phenylglyoxal, and it exhibits a preference for geranyl pyrophosphate over neryl pyrophosphate as substrate. An integrated scheme is proposed for the cyclization and rearrangement catalyzed by fenchol synthetase.