Molecular characterization and physiological role of ascorbate peroxidase from halotolerant Chlamydomonas sp. W80 strain.
A cDNA clone encoding an ascorbate peroxidase was isolated from the cDNA library from halotolerant Chlamydomonas W80 by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 282 amino acids with a calculated molecular mass of 30,031 Da, preceded by the chloroplast transit peptide consisting of 37 amino acids. In fact, ascorbate peroxidase was localized in the chloroplasts of Chlamydomonas W80 cells; the activity was detected in the stromal fraction but not in the thylakoid membrane. The deduced amino acid sequence of the cDNA showed 54 and 49% homology to chloroplastic and cytosolic ascorbate peroxidase isoenzymes of spinach leaves, respectively. The enzyme from Chlamydomonas W80 cells was purified to electrophoretic homogeneity. The molecular properties of the purified enzyme were similar to those of the other algal ascorbate peroxidases rather than those of ascorbate peroxidases from higher plants. The enzyme was relatively stable in ascorbate-depleted medium compared with the chloroplastic ascorbate peroxidase isoenzymes of higher plants. The presence of NaCl (3%) as well as of beta-d-thiogalactopyranoside was needed for the expression of Chlamydomonas W80 ascorbate peroxidase in Escherichia coli.
Takeda
T
T
Department of Food and Nutrition, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara, 631-8505, Japan.
Yoshimura
K
K
Yoshii
M
M
Kanahoshi
H
H
Miyasaka
H
H
Shigeoka
S
S
D000595:Amino Acid Sequence; D000818:Animals; D060387:Ascorbate Peroxidases; D001483:Base Sequence; D002696:Chlamydomonas / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017125:Genes, Protozoan; D007700:Kinetics; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012643:Selenium / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology
D017931:DNA Primers; D018076:DNA, Complementary; D016054:DNA, Protozoan; D011994:Recombinant Proteins; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D012643:Selenium