Heteromeric geranyl diphosphate synthase from mint: construction of a functional fusion protein and inhibition by bisphosphonate substrate analogs
Heteromeric geranyl diphosphate synthase from mint: construction of a functional fusion protein and inhibition by bisphosphonate substrate analogs
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DOI：
10.1016/j.abb.2003.12.003
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发表时间：
2004-02-01
影响因子：
3.9
通讯作者：
Croteau, R
中科院分区：
文献类型：
--
作者：
Burke, C;Klettke, K;Croteau, R
香叶基二磷酸合酶催化二甲基烯丙基二磷酸 (C-5) 与异戊烯基二磷酸 (C-5) 缩合，生成香叶基二磷酸 (C-10)，这是单萜的重要前体。来自薄荷和留兰香（分别为 Mentha x Piperita 和 Mentha spicata）的酶作为由 40 kDa 亚基和 33 kDa 亚基组成的异二聚体或异四聚体发挥作用。编码每个亚基的DNA与不同大小的接头以两种可能的顺序连接，并在大肠杆菌中表达以产生相应的融合蛋白。重组融合版本的特性（其中小亚基后面是带有 10 个氨基酸接头的大亚基）在动力学、产物链长度特异性和结构方面类似于天然异聚酶，因此这种形式为生物技术目的提供了合适的单基因转录本。抑制法尼基二磷酸合酶 (C-15) 和香叶基香叶基二磷酸合酶 (C-20) 类型的双膦酸酯底物类似物也抑制融合香叶基二磷酸合酶，显然是通过在烯丙基和同烯丙基共底物结合位点相互作用。抑制研究的结果以及先前确定的小亚基的作用和相关诱变实验表明，香叶基二磷酸合酶采用与其他短链异戊二烯基转移酶不同的机制来确定链长。 (C) 2003 Elsevier Inc. 保留所有权利。
Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate (C-5) with isopentenyl diphosphate (C-5) to produce geranyl diphosphate (C-10), the essential precursor of monoterpenes. The enzyme from peppermint and spearmint (Mentha x piperita and Mentha spicata, respectively) functions as a heterodimer or heterotetramer consisting of a 40 kDa subunit and 33 kDa subunit. The DNAs encoding each subunit were joined with different sized linkers and in both possible orders, and expressed in Escherichia coli to yield the corresponding fused protein. The properties of the recombinant fused version, in which the small subunit was followed by the large subunit with a 10 amino acid linker, resembled those of the native heteromeric enzyme in kinetics, product chain-length specificity, and architecture, and this form thus provided a suitable single gene transcript for biotechnological purposes. Bisphosphonate substrate analogs of the type that inhibit farnesyl diphosphate synthase (C-15) and geranylgeranyl diphosphate synthase (C-20) also inhibited the fused geranyl diphosphate synthase, apparently by interacting at both the allylic and homoallylic co-substrate binding sites. The results of inhibition studies, along with the previously established role of the small subunit and related mutagenesis experiments, suggest that geranyl diphosphate synthase employs a different mechanism for chain-length determination than do other short-chain prenyltransferases. (C) 2003 Elsevier Inc. All rights reserved.