INTRACELLULAR BETA-CARBONIC ANHYDRASE OF THE UNICELLULAR GREEN-ALGA COCCOMYXA

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible h ydration of CO,, a reaction that is important in many physiological pr ocesses. We have cloned and sequenced a full length cDNA encoding an i ntracellular P-CA from the unicellular green alga Coccomyxa. Nucleotid e sequence data show that the isolated cDNA contains an open reading f rame encoding a polypeptide of 227 amino acids. The predicted polypept ide is similar to beta-type CAs from Escherichia coli and higher plant s, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpresse d in E. coli, and the enzyme was purified and biochemically characteri zed. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Cocc omyxa CA is largely insensitive to oxidative conditions, in contrast t o similar enzymes from most higher plants. Fractionation studies furth er showed that Coccomyxa CA is extrachloroplastic.