Molecular cloning and characterization of a protein farnesyltransferase from the enteric protozoan parasite Entamoeba histolytica
Molecular cloning and characterization of a protein farnesyltransferase from the enteric protozoan parasite Entamoeba histolytica
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DOI：
10.1074/jbc.m311478200
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发表时间：
2004-01-16
影响因子：
4.8
通讯作者：
Nozaki, T
中科院分区：
文献类型：
--
作者：
Kumagai, M;Makioka, A;Nozaki, T
从肠道原生动物寄生虫溶组织内阿米巴中获得了编码推定蛋白法尼基转移酶（FT）的α-和β-亚基的基因，并对其生化特性进行了表征。推导了E.溶组织菌FT（EhFT）长298和375个残基，分子量分别为35.6和42.6kDa，pI分别为5.43和5.65。它们与来自其他生物体的序列显示出24%至36%的同一性，并具有共同的特征结构域和重复序列。重组α-和β-亚基，在大肠杆菌中共表达，形成异源二聚体，并显示出使用法呢焦磷酸作为供体转移法呢基到具有C-末端CVLS的人H-Ras的活性，但不是具有CVLL的突变体H-Ras。在许多属于Ras超家族的小GTP酶中，我们鉴定了EhRas4，其在C末端具有CVVA，作为EhFT的唯一法尼基受体。这与哺乳动物FT相反，哺乳动物FT利用具有C-末端CaaX基序的多种小GTP酶，其中X是丝氨酸、甲硫氨酸、谷氨酰胺、半胱氨酸或丙氨酸。EhFT还显示出对多种已知哺乳动物FT抑制剂的显著抗性。这些结果表明，显着的生物化学差异，结合到基板和抑制剂之间存在阿米巴和哺乳动物的FT，这突出了这种酶作为一个新的目标，开发新的化学治疗阿米巴病。
Genes encoding alpha- and beta-subunits of a putative protein farnesyltransferase (FT) from the enteric protozoan parasite Entamoeba histolytica were obtained and their biochemical properties were characterized. Deduced amino acid sequences of the alpha- and beta-subunit of E. histolytica FT (EhFT) were 298- and 375-residues long with a molecular mass of 35.6 and 42.6 kDa, and a pI of 5.43 and 5.65, respectively. They showed 24% to 36% identity to and shared common signature domains and repeats with those from other organisms. Recombinant alpha- and beta-subunits, co-expressed in Escherichia coli, formed a heterodimer and showed activity to transfer farnesyl using farnesylpyrophosphate as a donor to human H-Ras possessing a C-terminal CVLS, but not a mutant H-Ras possessing CVLL. Among a number of small GTPases that belong to the Ras superfamily from this parasite, we identified EhRas4, which possesses CVVA at the C terminus, as a sole farnesyl acceptor for EhFT. This is in contrast to mammalian FT, which utilizes a variety of small GTPases that possess a C-terminal CaaX motif, where X is serine, methionine, glutamine, cysteine, or alanine. EhFT also showed remarkable resistance against a variety of known inhibitors of mammalian FT. These results suggest that remarkable biochemical differences in binding to substrates and inhibitors exist between amebic and mammalian FTs, which highlights this enzyme as a novel target for the development of new chemotherapeutics against amebiasis.