Identification and characterization of two closely related β‐carbonic anhydrases from Chlamydomonas reinhardtii

Abstract

Aquatic photosynthetic organisms such as the green alga Chlamydomonas reinhardtii respond to low-CO(2) conditions by inducing a CO(2) concentrating mechanism (CCM). Important components of the CCM are the carbonic anhydrases (CAs), zinc metalloenzymes that catalyze the interconversion of CO(2) and HCO(-)(3). Six CAs have previously been identified in C. reinhardtii. Here, we identify and characterize two additional beta-type CAs. These two CAs are closely related beta-type CAs and have been designated as CAH7 and CAH8. Conceptual translation shows that CAH7 and CAH8 encode proteins of 399 and 333 amino acids, respectively, and they contain targeting sequences. An unusual characteristic of these two CAs is that they have carboxy-terminal extensions containing a hydrophobic sequence. Both these CAs are constitutively expressed at the transcript and protein level. The CAH7 and CAH8 open reading frames were cloned in the overexpression vector pMal-c2x and expressed as recombinant proteins. Activity assays showed that CAH7 and CAH8 are both active CAs. Antibodies were raised against both CAH7 and CAH8, and immunolocalization studies showed that CAH8 was localized in the periplasmic space. A possible role for CAH8 in the inorganic carbon acquisition by C. reinhardtii is discussed.