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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Molecular and Biochemical Characterization of the Parvulin-Type PPIases in Lotus japonicus^{1,[C],[W],[OA]}

Laboratory of Molecular Biology (E.D.K., K.I.K., C.S., M.D., P.K., E.F.) and Laboratory of Enzyme Technology (N.E.L.), Department of Agricultural Biotechnology, Agricultural University of Athens, 11855 Athens, Greece; Center for Basic Research, Biomedical Research Foundation of the Academy of Athens, 11527 Athens, Greece (S.D.G.); and Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, Oklahoma 73401 (M.K.U.)

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.

² Present address: Laboratory of Molecular Virology, Hellenic Pasteur Institute, Vasilissis Sofias 127, 11521 Athens, Greece.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Emmanouil Flemetakis (mflem{at}aua.gr).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[W] The online version of this article contains Web-only data.

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www.plantphysiol.org/cgi/doi/10.1104/pp.108.132415

^* Corresponding author; e-mail mflem{at}aua.gr.

Received November 11, 2008; accepted April 26, 2009; published April 29, 2009.

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