title: Euglena gracilis ascorbate peroxidase forms an intramolecular dimeric structure: its unique molecular characterization.
abstract: Euglena gracilis lacks a catalase and contains a single APX (ascorbate peroxidase) and enzymes related to the redox cycle of ascorbate in the cytosol. In the present study, a full-length cDNA clone encoding the Euglena APX was isolated and found to contain an open reading frame encoding a protein of 649 amino acids with a calculated molecular mass of 70.5 kDa. Interestingly, the enzyme consisted of two entirely homologous catalytic domains, designated APX-N and APX-C, and an 102 amino acid extension in the N-terminal region, which had a typical class II signal proposed for plastid targeting in Euglena. A computer-assisted analysis indicated a novel protein structure with an intramolecular dimeric structure. The analysis of cell fractionation showed that the APX protein is distributed in the cytosol, but not the plastids, suggesting that Euglena APX becomes mature in the cytosol after processing of the precursor. The kinetics of the recombinant mature FL (full-length)-APX and the APX-N and APX-C domains with ascorbate and H2O2 were almost the same as that of the native enzyme. However, the substrate specificity of the mature FL-APX and the native enzyme was different from that of APX-N and APX-C. The mature FL-APX, but not the truncated forms, could reduce alkyl hydroperoxides, suggesting that the dimeric structure is correlated with substrate recognition. In Euglena cells transfected with double-stranded RNA, the silencing of APX expression resulted in a significant increase in the cellular level of H2O2, indicating the physiological importance of APX to the metabolism of H2O2.
authors.affiliation: Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan. ishikawa@life.shimane-u.ac.jp
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Naoko
authors.initials: N
authors.lastname: Tajima
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nishikawa
authors.forename: Yongshun
authors.initials: Y
authors.lastname: Gao
authors.forename: Madhusudhan
authors.initials: M
authors.lastname: Rapolu
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shibata
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sawa
authors.forename: Shigeru
authors.initials: S
authors.lastname: Shigeoka
mesh_terms: D000595:Amino Acid Sequence; D060387:Ascorbate Peroxidases; D003600:Cytosol / Q000201:enzymology; D019281:Dimerization; D005056:Euglena gracilis / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D015800:Protozoan Proteins; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases