title: Stable form of ascorbate peroxidase from the red alga Galdieria partita similar to both chloroplastic and cytosolic isoforms of higher plants.
abstract: Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, Km values, kcat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured. Recombinant APX-B remained active for at least 180 min after depletion of ascorbate. The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs. In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform. The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.
authors.affiliation: Research Institute of Innovative Technology for the Earth (RITE), Soraku-gun, Kyoto, 619-0292, Japan.
authors.forename: Sakihito
authors.initials: S
authors.lastname: Kitajima
authors.forename: Masami
authors.initials: M
authors.lastname: Ueda
authors.forename: Satoshi
authors.initials: S
authors.lastname: Sano
authors.forename: Chikahiro
authors.initials: C
authors.lastname: Miyake
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kohchi
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Tomizawa
authors.forename: Shigeru
authors.initials: S
authors.lastname: Shigeoka
authors.forename: Akiho
authors.initials: A
authors.lastname: Yokota
mesh_terms: D000595:Amino Acid Sequence; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D000460:Chlorophyta / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology*; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D010944:Plants / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000461:Rhodophyta / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid