Vogel B S, Wildung M R, Vogel G, Croteau R
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
J Biol Chem. 1996 Sep 20;271(38):23262-8. doi: 10.1074/jbc.271.38.23262.
(-)-Abietic acid, the principal diterpenoid resin acid of the wound-induced oleoresin secreted by grand fir (Abies grandis), is synthesized by the cyclization of geranylgeranyl diphosphate to (-)-abieta-7(8),13(14)-diene, followed by sequential three-step oxidation of the C-18 methyl group of the olefin to a carboxyl function. The enzyme catalyzing the cyclization reaction, abietadiene synthase, was purified from stems of wounded grand fir saplings and was digested with trypsin. Amino acid sequence information from the resulting peptides allowed construction of degenerate oligonucleotide primers, which amplified a 551-base pair fragment from a wound-induced stem cDNA library. This hybridization probe was then utilized to screen the wound-induced stem cDNA library, from which three cDNA clones were isolated that were functionally expressed in Escherichia coli, thereby confirming that a single protein catalyzes the complex, multistep cyclization of geranylgeranyl diphosphate to abietadiene. cDNA isolate Ac22.1, which yielded the highest expressed level of cyclase activity, was 2861 base pairs in length and encoded an 868-amino acid open reading frame that included a putative plastidial transit peptide. Deduced amino acid sequence comparison to other terpene cyclases revealed an amino-terminal region of the abietadiene synthase, which resembles those of enzymes that employ substrate double bond protonation to initiate the carbocationic reaction cascade, and a carboxyl-terminal region of the synthase, which resembles those of enzymes that employ ionization of the substrate allylic diphosphate ester function to initiate the cyclization reaction. This apparent fusion of segments of the two distinct terpenoid cyclase types is consistent with the novel mechanism of the bifunctional abietadiene synthase in catalyzing both protonation-initiated and ionization-initiated cyclization steps.
（-）-枞酸是大冷杉（Abies grandis）伤口诱导分泌的油性树脂中的主要二萜树脂酸，它由香叶基香叶基二磷酸环化生成（-）-枞-7（8），13（14）-二烯，随后烯烃的C-18甲基依次进行三步氧化生成羧基官能团。催化环化反应的酶——枞二烯合酶，从受伤的大冷杉幼树茎中纯化出来并用胰蛋白酶消化。从所得肽段获得的氨基酸序列信息使得能够构建简并寡核苷酸引物，该引物从伤口诱导的茎cDNA文库中扩增出一个551个碱基对的片段。然后利用该杂交探针筛选伤口诱导的茎cDNA文库，从中分离出三个在大肠杆菌中具有功能表达的cDNA克隆，从而证实单一蛋白质催化香叶基香叶基二磷酸向枞二烯的复杂多步环化反应。cDNA分离物Ac22.1产生的环化酶活性表达水平最高，长度为2861个碱基对，编码一个868个氨基酸的开放阅读框，其中包括一个推定的质体转运肽。将推导的氨基酸序列与其他萜烯环化酶进行比较，发现枞二烯合酶的氨基末端区域类似于那些利用底物双键质子化启动碳正离子反应级联的酶，而合酶的羧基末端区域类似于那些利用底物烯丙基二磷酸酯官能团的电离启动环化反应的酶。这两种不同萜类环化酶类型片段的明显融合与双功能枞二烯合酶催化质子化引发和电离引发环化步骤的新机制一致。
