Kouri Evangelia D, Labrou Nikolaos E, Garbis Spiros D, Kalliampakou Katerina I, Stedel Catalina, Dimou Maria, Udvardi Michael K, Katinakis Panagiotis, Flemetakis Emmanouil
Laboratory of Molecular Biology, Department of Agricultural Biotechnology, Agricultural University of Athens, 11855 Athens, Greece.
Plant Physiol. 2009 Jul;150(3):1160-73. doi: 10.1104/pp.108.132415. Epub 2009 Apr 29.
The cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, although important in many biological processes in both prokaryotes and eukaryotes. In vivo, this isomerization is catalyzed by peptidyl-prolyl cis/trans-isomerases (PPIases). Here, we present the molecular and biochemical characterization of parvulin-type PPIase family members of the model legume Lotus japonicus, annotated as LjPar1, LjPar2, and LjPar3. Although LjPar1 and LjPar2 were found to be homologous to PIN1 (Protein Interacting with NIMA)-type parvulins and hPar14 from human, respectively, LjPar3 represents a novel multidomain parvulin, apparently present only in plants, that contains an active carboxyl-terminal sulfurtransferase domain. All Lotus parvulins were heterologously expressed and purified from Escherichia coli, and purified protein verification measurements used a liquid chromatography-mass spectrometry-based proteomic method. The biochemical characterization of the recombinant Lotus parvulins revealed that they possess PPIase activity toward synthetic tetrapeptides, although they exhibited different substrate specificities depending on the amino acid amino terminal to proline. These differences were also studied in a structural context using molecular modeling of the encoded polypeptides. Real-time reverse transcription-polymerase chain reaction revealed that the three parvulin genes of Lotus are ubiquitously expressed in all plant organs. LjPar1 was found to be up-regulated during the later stages of nodule development. Subcellular localization of LjPar-enhanced Yellow Fluorescence Protein (eYFP) fusions expressed in Arabidopsis (Arabidopsis thaliana) leaf epidermal cells revealed that LjPar1- and LjPar2-eYFP fusions were localized in the cytoplasm and in the nucleus, in contrast to LjPar3-eYFP, which was clearly localized in plastids. Divergent substrate specificities, expression profiles, and subcellular localization indicate that plant parvulin-type PPIases are probably involved in a wide range of biochemical and physiological processes.
脯氨酸之前的肽键顺反异构化是一个本质上缓慢的过程，尽管在原核生物和真核生物的许多生物学过程中都很重要。在体内，这种异构化由肽基脯氨酰顺反异构酶（PPIase）催化。在这里，我们展示了模式豆科植物百脉根中细小菌素型PPIase家族成员的分子和生化特征，分别注释为LjPar1、LjPar2和LjPar3。虽然发现LjPar1和LjPar2分别与人的PIN1（与NIMA相互作用的蛋白质）型细小菌素和hPar14同源，但LjPar3代表一种新型的多结构域细小菌素，显然仅存在于植物中，其含有一个活性羧基末端硫转移酶结构域。所有百脉根细小菌素都在大肠杆菌中进行了异源表达和纯化，纯化蛋白验证测量采用了基于液相色谱 - 质谱的蛋白质组学方法。重组百脉根细小菌素的生化特征表明，它们对合成四肽具有PPIase活性，尽管根据脯氨酸氨基末端的氨基酸不同，它们表现出不同的底物特异性。还使用编码多肽的分子模型在结构背景下研究了这些差异。实时逆转录 - 聚合酶链反应表明，百脉根的三个细小菌素基因在所有植物器官中均普遍表达。发现LjPar1在根瘤发育后期上调。在拟南芥叶表皮细胞中表达的LjPar - 增强型黄色荧光蛋白（eYFP）融合蛋白的亚细胞定位表明，LjPar1 - eYFP和LjPar2 - eYFP融合蛋白定位于细胞质和细胞核中，而LjPar3 - eYFP则明显定位于质体中。不同的底物特异性、表达谱和亚细胞定位表明，植物细小菌素型PPIase可能参与广泛的生化和生理过程。
