Identification of a novel class of ω,E,E-farnesyl diphosphate synthase from Mycobacterium tuberculosis
Identification of a novel class of ω,E,E-farnesyl diphosphate synthase from Mycobacterium tuberculosis
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DOI：
10.1194/jlr.m400047-jlr200
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发表时间：
2004-06-01
影响因子：
6.5
通讯作者：
Crick, DC
中科院分区：
文献类型：
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作者：
Dhiman, RK;Schulbach, MC;Crick, DC
我们已经从结核分枝杆菌中鉴定了由开放阅读框Rv 3398 c编码的ω，E，E-法呢基二磷酸（ω，E，E-FPP）合酶，其在报道的FPP合酶中是独特的，因为它不包含I型（真核）或II型（真细菌）ω，E，E-FPP合酶特征基序。相反，它有一个结构基序类似的I型香叶基香叶基二磷酸合成酶中发现的美洲。因此，该酶代表了一类新的ω，E，E-FPP合酶。Rv 3398 c是从M.结核病H37 Rv基因组中，并使用新的分枝杆菌表达载体（pVV 2）在耻垢分枝杆菌中表达，所述新的分枝杆菌表达载体编码与目的蛋白质的框内N-末端亲和标签融合。融合蛋白表达良好，并可纯化至接近同质，从而允许重组Rv 3398 c的容易的动力学分析。在测试的潜在烯丙基底物中，包括二甲基烯丙基二磷酸，只有香叶基二磷酸充当异戊烯基二磷酸的受体。该酶对二价阳离子有绝对需求，对异戊烯基二磷酸的Km为43 μ M，对香叶基二磷酸的Km为9.8 μ M，据报道该酶对M的生存力是必需的。结核
We have identified an omega,E,E-farnesyl diphosphate (omega,E,E-FPP) synthase, encoded by the open reading frame Rv3398c, from Mycobacterium tuberculosis that is unique among reported FPP synthases in that it does not contain the type I (eukaryotic) or the type II (eubacterial) omega,E,E- FPP synthase signature motif. Instead, it has a structural motif similar to that of the type I geranylgeranyl diphosphate synthase found in Archaea. Thus, the enzyme represents a novel class of omega,E,E-FPP synthase. Rv3398c was cloned from the M. tuberculosis H37Rv genome and expressed in Mycobacterium smegmatis using a new mycobacterial expression vector (pVV2) that encodes an in-frame N-terminal affinity tag fusion with the protein of interest. The fusion protein was well expressed and could be purified to near homogeneity, allowing facile kinetic analysis of recombinant Rv3398c. Of the potential allylic substrates tested, including dimethylallyl diphosphate, only geranyl diphosphate served as an acceptor for isopentenyl diphosphate. The enzyme has an absolute requirement for divalent cation and has a K-m of 43 muM for isopentenyl diphosphate and 9.8 muM for geranyl diphosphate and is reported to be essential for the viability of M. tuberculosis.