Identification, Cloning and Characterization of Two Closely Related β‐Carbonic Anhydrases in Chlamydomonas reinhardtii

Abstract

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that are involved in important biochemical reactions such as photosynthesis and respiration. For example, CAs play important roles in the Carbon Concentrating Mechanism (CCM) present in aquatic photosynthetic organisms such as Chlamydomonas reinhardtii. The presence of the CCM enables these organisms to overcome the slow diffusion of CO2 in water and thereby photosynthesize efficiently. Recently we have identified genes encoding two closely related β-type CAs. This brings the number of CA genes in C. reinhardtii to eight and these new CAs have been designated Cah7 and Cah8. Nucleotide sequence data show that Cah7 cDNA encodes a protein of 399 amino acids while Cah8 cDNA encodes a protein of 354 amino acids. Cah7 and Cah8 have an identity of 74%. These proteins are predicted to be targeted either to the cytosol or chloroplast stroma. We have overexpressed these two CAs in Escherichia coli to study their properties and raise antibodies. Determination of CA activity demonstrated that both Cah7 and Cah8 encode active CAs. We have already raised an antibody to Cah8 and doing immunolocalization of Cah8. Expression analyses show that these newly identified CAs are constitutively expressed under both high CO2 and low CO2 conditions. RNAi constructs been generated to further elucidate the possible roles of Cah7 and Cah8 in C. reinhardtii. This study is supported by the National Science Foundation.