Stable form of ascorbate peroxidase from the red alga Galdieria partita similar to both chloroplastic and cytosolic isoforms of higher plants.
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.
Kitajima
Sakihito
S
Research Institute of Innovative Technology for the Earth (RITE), Soraku-gun, Kyoto, 619-0292, Japan.
Ueda
Masami
M
Sano
Satoshi
S
Miyake
Chikahiro
C
Kohchi
Takayuki
T
Tomizawa
Ken-ichi
K
Shigeoka
Shigeru
S
Yokota
Akiho
A
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
D018076:DNA, Complementary; D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid