Germacrene A is a product of the aristolochene synthase-mediated conversion of farnesylpyrophosphate to aristolochene
Germacrene A is a product of the aristolochene synthase-mediated conversion of farnesylpyrophosphate to aristolochene
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DOI：
10.1021/ja020762p
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发表时间：
2002-10-02
影响因子：
15
通讯作者：
Allemann, RK
中科院分区：
文献类型：
--
作者：
Calvert, MJ;Ashton, PR;Allemann, RK
几种倍半萜的生物合成已被提出通过大根香叶烯A进行。然而，到目前为止，对于任何倍半萜烯脱氢酶，都没有直接证明大根香叶烯A的产生，而大根香叶烯A被假定为所述倍半萜烯脱氢酶的中间体。我们在这里首次证明，大量的吉马烯A（占产物总量的7.5%）确实从罗克福特青霉的野生型马兜铃烯合酶（AS）中释放出来。通过与真实样品的直接GC-MS比较以及通过热科普重排中β-榄香烯的产生来鉴定大根香叶烯A。AS还通过在反应的最后步骤中C6而不是C8的去质子化产生少量朱栾倍半萜。根据AS的X-射线结构，推测Tyr 92是负责大根香叶烯A质子化的活性位点酸（卡鲁瑟斯，J.M.; Kang，L; Rynkiewicz，M. J.道：Cane，D. E.的; Christianson，D. W. 2000，275，25533-25539）。其中Tyr 92被Phe取代的突变蛋白ASY 92 F的CID谱与AS的CID谱非常相似。ASY 92 F的活性约为非突变重组AS的0.1%。k（cat）和K-M的稳态动力学参数分别为0.138 min（-1）和0.189 mM。类似于5-表马兜铃烯的突变蛋白（Rising，K.一、斯塔克斯角M.;诺埃尔，J. P.;查普尔，J. J. Am. Soc.2000，122，1861 - 1866），该突变体释放显著量的大根香叶烯A（类似于29%）。ASY 92 F还产生了不同量的另外五种分子量为204的烃，朱栾倍半萜、β-（E）-法呢烯、α-和β-芹烯以及芹烯-4，11-二烯。
The biosynthesis of several sesquiterpenes has been proposed to proceed via germacrene A. However, to date, the production of germacrene A has not been proven directly for any of the sesquiterpene synthases for which it was postulated as an intermediate. We demonstrate here for the first time that significant amounts of germacrene A (7.5% of the total amount of products) are indeed released from wild-type aristolochene synthase (AS) from Penicillium roqueforti. Germacrene A was identified through direct GC-MS comparison to an authentic sample and through production of beta-elemene in a thermal Cope rearrangement. AS also produced a small amount of valencene through deprotonation of C6 rather than C8 in the final step of the reaction. On the basis of the X-ray structure of AS, Tyr 92 was postulated to be the active-site acid responsible for protonation of germacrene A (Caruthers, J. M.; Kang, L; Rynkiewicz, M. J.; Cane, D. E.; Christianson, D. W. J. Biol. Chem. 2000, 275, 25533-25539). The CID spectra of a mutant protein, ASY92F, in which Tyr 92 was replaced by Phe, and of AS were very similar. ASY92F was approximately 0.1% as active as nonmutated recombinant AS. The steady-state kinetic parameters were measured as 0.138 min(-1) and 0.189 mM for k(cat) and K-M, respectively. Similar to a mutant protein of 5-epiaristolochene (Rising, K. A.; Starks, C. M.; Noel, J. P.; Chappell, J. J. Am. Chem. Soc. 2000, 122, 1861 1866), the mutant released significant amounts of germacrene A (similar to29%). ASY92F also produced various amounts of a further five hydrocarbons of molecular weight 204, valencene, beta-(E)-farnesene, alpha- and beta-selinene, and selina-4,11-diene.