Molecular identification, heterologous expression and properties of light-insensitive plant catalases.
Most catalases are inactivated by light in a heme-sensitized and O2-dependent reaction. In leaves of the alpine plant Homogyne alpina and in the peroxisomal cores of Helianthus annuus, light-insensitive catalases were observed. For the catalases Hacat1 of H. alpina and HnncatA3 of H. annuus, cDNA clones were obtained. Expression of recombinant active enzymes in insect cells confirmed that they coded for light-insensitive catalases. Kinetic and catalytic properties of light-sensitive or light-insensitive catalases did not differ substantially. However, the specific activity of the latter was markedly lower. The light-insensitive catalase HaCAT-1 was not resistant against inactivation by superoxide. Amino acid sequences of the light-insensitive catalases HaCAT-1 and HNNCATA3 were highly identical. They showed only a few exceptional amino acid substitutions at positions that are highly conserved in other catalases. These appeared to be localized mainly in a surface cavity at the entrance of a minor channel leading to the central heme, suggesting that this region played some, though yet undefined, role for light sensitivity. While the replacement of a highly conserved His by Thr225 was the most unique substitution, a single exchange of His225 by Thr in the light-sensitive catalase SaCAT-1 by mutagenesis was not sufficient to reduce its sensitivity to photoinactivation.
Engel
Nicole
N
Botanisches Institut, Goethe-Universität, D-60054 Frankfurt am Main, Germany.
Schmidt
Matthias
M
Lütz
Cornelius
C
Feierabend
Jürgen
J
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D019659:Asteraceae / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D002374:Catalase / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D003001:Cloning, Molecular; D006368:Helianthus / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000528:radiation effects; D020675:Peroxisomes / Q000201:enzymology / Q000648:ultrastructure; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012434:Secale / Q000201:enzymology; D016415:Sequence Alignment; D018411:Spodoptera / Q000235:genetics; D013481:Superoxides / Q000494:pharmacology
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013481:Superoxides; D002374:Catalase