Tholl Dorothea, Kish Christine M, Orlova Irina, Sherman Debra, Gershenzon Jonathan, Pichersky Eran, Dudareva Natalia
Max Planck Institute for Chemical Ecology, D-007745 Jena, Germany.
Plant Cell. 2004 Apr;16(4):977-92. doi: 10.1105/tpc.020156. Epub 2004 Mar 18.
The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit (GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU, might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.
所有单萜类化合物的前体是C10无环中间体香叶基二磷酸（GPP），它由C5化合物异戊烯基二磷酸和二甲基烯丙基二磷酸通过GPP合酶（GPPS）形成。我们发现，金鱼草和布鲁氏克拉克花这两种花香富含单萜类化合物的植物，都拥有一种异源二聚体GPPS，类似于先前从薄荷中报道的那种。金鱼草和布鲁氏克拉克花的cDNA分别编码与薄荷GPPS小亚基（GPPS.SSU）氨基酸序列同一性为53%和45%的蛋白质。这些cDNA在大肠杆菌中表达未产生可检测到的异戊烯基转移酶活性。然而，当这些cDNA中的每一个与薄荷GPPS大亚基（GPPS.LSU）共表达时，可获得活性GPPS，薄荷GPPS大亚基与香叶基香叶基二磷酸合酶（GGPPS）具有功能基序和高水平的氨基酸序列同一性。使用基于同源性的克隆策略，还从金鱼草中分离出了GPPS.LSU cDNA。它在大肠杆菌中与金鱼草GPPS.SSU共表达产生了一种功能性异源二聚体，该异源二聚体催化以GPP为主要产物的合成。金鱼草GPPS.LSU在大肠杆菌中单独表达产生了活性GGPPS，这表明与薄荷GPPS.LSU不同，金鱼草GPPS.LSU本身就是一种功能性GGPPS。对金鱼草花朵中GPPS.SSU的mRNA和蛋白质水平的组织特异性、发育性和节律性变化分析表明，这些水平与单萜类化合物的释放密切相关，而GPPS.LSU的mRNA水平则不然，这表明GPPS.SSU的水平而非GPPS.LSU的水平可能在调节GPPS的形成以及单萜类化合物生物合成中起关键作用。
