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Identification of a new chloroplast carbonic anhydrase in Chlamydomonas reinhardtii

Mitra, M., Lato, S.M., Ynalvez, R.A., Xiao, Y., Moroney, J.V.
Plant physiology 2004 v.135 no.1 pp. 173-182
Chlamydomonas reinhardtii, algae and seaweeds, chloroplasts, plant proteins, carbonate dehydratase, complementary DNA, DNA libraries, genomics, gene overexpression, genetic vectors, recombinant fusion proteins, purification, enzyme activity, gene expression regulation, Northern blotting, sequence analysis, sequence alignment, amino acid sequences, nucleotide sequences, Western blotting
Carbonic anhydrases (CA) are zinc-containing metalloenzymes that catalyze the reversible hydration of CO2. The three evolutionarily unrelated families of CAs are designated alpha-, beta-, and gamma-CA. Aquatic photosynthetic organisms have evolved different forms of CO2 concentrating mechanisms (CCMs) to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CCMs is the critical roles played by various specially localized extracellular and intracellular CAs. Five CAs have previously been identified in Chlamydomonas reinhardtii, a green alga with a well-studied CCM. Her e we identify a sixth gene encoding a beta-type CA. This new beta-CA, designated Cah6, is d istinct from the two mitochondrial beta-CAs in C. reinhardtii. Nucleotide sequence data show that the Cah6 cDNA contains an open reading frame encoding a polypeptide of 264 amino acids with a leader sequence likely targeting the protein to the chloroplast stroma. We have fused the Cah6 open reading frame to the coding sequence of maltose-binding protein in a pMal expression vector. The purified recombinant fusion protein is active and was used to partially characterize the Cah6 protein. The purified recombinant fusion protein was cleaved with protease Factor Xa to separate Cah6 from the maltose-binding protein and the purified Cah6 protein was used to raise an antibody. Western blots, immunolocalization studies, and northern blots collectively indicated that Cah6 is constitutively expressed in the stroma of chloroplasts. A possible role for Cah6 in the CCM of C. reinhardtii is proposed.