Hefner J, Ketchum R E, Croteau R
Institute of Biological Chemistry, and Department of Genetics and Cell Biology, Washington State University, Pullman, Washington, 99164-6340, USA.
Arch Biochem Biophys. 1998 Dec 1;360(1):62-74. doi: 10.1006/abbi.1998.0926.
Geranylgeranyl diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-induced Taxus canadensis suspension cell cultures. A cDNA encoding this prenyltransferase was cloned from an induced T. canadensis cell library. The recombinant enzyme expressed in yeast was confirmed by radiochromatographic analysis to produce geranylgeranyl diphosphate from farnesyl diphosphate and [4-14C]isopentenyl diphosphate and was subjected to preliminary kinetic characterization. The deduced amino acid sequence of this gymnosperm geranylgeranyl diphosphate synthase (393 residues) resembles those of geranylgeranyl diphosphate synthases of angiosperm origin, except for the 90-100 N-terminal residues that correspond to the plastidial transit peptide. The full-length preprotein (42.6 kDa) and two truncated versions, corresponding to putative "mature proteins" from which the transit peptide was deleted, were transformed into a yeast mutant defective for the beta-subunit of type II geranylgeranyl transferase. Under conditions of regulated expression, both the full-length construct and the longest of the truncations (at Phe 99) were able to complement the mutant. However, when these two constructs were overexpressed in a wild-type yeast strain, they were apparently toxic, most probably due to depletion of endogenous farnesyl diphosphate as the cosubstrate for the geranylgeranyl diphosphate synthase reaction. In vitro activity of the corresponding recombinant enzymes paralleled the expression level of the constructs as determined by SDS-PAGE analysis of the appropriate proteins of predicted size, and was correlated with toxicity in the wild-type yeast strain and with ability to complement the mutant strain. Results from the analysis of geranylgeranyl diphosphate synthase activity levels and measurement of the corresponding steady-state mRNA levels during the time course of Taxol production in induced T. canadensis suspension cell cultures, and comparison to similar data for activity and message levels for taxadiene synthase, the committed step of the pathway, indicated that for each enzyme both the level of corresponding message and catalytic activity rapidly increased after methyl jasmonate induction.
香叶基香叶基二磷酸合酶为茉莉酸甲酯诱导的加拿大红豆杉悬浮细胞培养物中紫杉醇生物合成提供必需的无环前体。从诱导的加拿大红豆杉细胞文库中克隆了编码该异戊二烯基转移酶的cDNA。通过放射色谱分析证实，在酵母中表达的重组酶能从法尼基二磷酸和[4-¹⁴C]异戊烯基二磷酸生成香叶基香叶基二磷酸，并对其进行了初步的动力学表征。这种裸子植物香叶基香叶基二磷酸合酶推导的氨基酸序列（393个残基）与被子植物来源的香叶基香叶基二磷酸合酶相似，除了对应于质体转运肽的90-100个N端残基。全长前体蛋白（42.6 kDa）和两个截短版本，对应于缺失转运肽的假定“成熟蛋白”，被转化到II型香叶基香叶基转移酶β亚基缺陷的酵母突变体中。在调控表达条件下，全长构建体和最长的截短体（在苯丙氨酸99处）都能够互补突变体。然而，当这两种构建体在野生型酵母菌株中过表达时，它们显然具有毒性，最可能的原因是作为香叶基香叶基二磷酸合酶反应共底物的内源性法尼基二磷酸被耗尽。相应重组酶的体外活性与构建体的表达水平平行，这通过对预测大小的适当蛋白质进行SDS-PAGE分析来确定，并且与野生型酵母菌株中的毒性以及互补突变体菌株的能力相关。在诱导的加拿大红豆杉悬浮细胞培养物中紫杉醇产生的时间进程中，香叶基香叶基二磷酸合酶活性水平分析结果以及相应稳态mRNA水平的测量结果，与该途径的关键步骤紫杉二烯合酶的活性和信息水平的类似数据进行比较，表明对于每种酶，相应信息的水平和催化活性在茉莉酸甲酯诱导后迅速增加。
