Cytosolic isoform-like structure of domain II is important for the stability of ascorbate peroxidase of Galdieria partita under ascorbate-depleted conditions.

Abstract

Chloroplastic ascorbate peroxidases (APXs), enzymes involved in scavenging system of reactive oxygen species, are rapidly inactivated by attack of hydrogen peroxide if the electron donor ascorbate is not available. In contrast, another APX isoform localized in cytosol is relatively stable. The mechanism of the different stability among APX isoforms remains unknown. We isolated a clone of the cDNA encoding a stable isoform of ascorbate peroxidase (APX-B) of Galdieria partita, an acido- and thermophilic red alga. The recombinant protein of APX-B produced in Escherichia coli retained its activity when it was incubated in the medium containing 10 mM of ascorbate for 3 hr, while recombinant stromal APX of spinach chloroplasts lost its activity within 1 hr due to the depletion of ascorbate. Interestingly, APX-B was a hybrid between the cytosolic and chloroplastic APXs in its primary structure; the N-terminal half (domain I) relatively similar to that of domain I of the chloroplastic isoform of higher plants and the C-terminal half (domain II) similar to that of domain II of the cytosolic isoform. A chimeric APX protein that had a domain I of APX-B fused to a domain II of stromal APX of spinach was created. It showed rapid inactivation when ascorbate was depleted. These results indicate that cytosolic APX-like domain II is important for its stability of Galdieria APX-B and the stability or instability of APX isoforms is due to their structures of domain II, rather than domain I.