Isolation and Characterisation od a Thioredoxin-dependent peroxidase in Chlamydomomnas reinhardtii.

Abstract

Living organisms have developped redox signaling systems involving small proteins named thioredoxins (Trx) featuring an extremely conserved and reactive active site WC(G)PC that performs thiol-disulfide interchanges with disulfide bridges of various target proteins. In photosynthetic organisms, many functions of Trx are most likely still to be uncovered, since sequencing of Arabidopsis thaliana and Chlamydomonas reinhardtii genomes revealed numerous Trx isoforms. In Chlamydomonas, 4 putative cytosolic (h-type), and 4 chloroplastic (2 f-type and 2 m-type) isoforms were identified. In an attempt to find new molecular targets of Trx in Chlamydomonas reinhardtii, a technique based on the reaction mechanism of these proteins, i.e. transient formation of mixed disulfides with their target, was used. An affinity column made of a Trx h mutated at the less reactive cysteine of its active site allowed to trap a major protein, identified by amino-acid sequencing as a thioredoxin-dependent, probably chloroplastic, peroxidase. We characterized its antioxidant properties against reactive oxygen species (ROS) and its peroxidase activity. After cloning the cDNA and gene, we studied the regulation of its expression under different physiological conditions. A strong increase in expression under high oxygen concentration was observed, suggesting a possible role of thioredoxins in ROS detoxification.