Characterization of a novel delta class carbonic anhydrase and its role in carbon metabolism in the marine alga Emiliania huxleyi

The marine alga, Emiliania huxleyi has ability to synthesize unique sculptured\ud calcium carbonate structures, called coccoliths, and has attracted the attention of\ud scientists interested in topics ranging from marine ecology to biomedical materials\ud science. Growth of E. huxleyi is strictly photoautotrophic and the key enzyme\ud involved in the carbon metabolism and calcification in microalgae is carbonic\ud anhydrase (CA). Until recently, the research approach in this organism focused on\ud the partitioning of inorganic carbon between calcification and photosynthesis,\ud primarily using measurements of an external versus internal carbonic anhydrase\ud activity under defined conditions. The actual genes, proteins, and pathways\ud employed in these processes have not been identified and characterized. In this\ud thesis, the cloning and preliminary characterization of a novel delta class carbonic\ud anhydrase (b-EhCAl) is described; this is the first gene and protein involved in\ud carbon metabolism to be isolated and characterized in coccolithophorid algae. The\ud recombinant protein was expressed in E. coli and the subcloned CA-region (329 aa)\ud of the protein showed significant enzyme activity. Western blot analyses indicated\ud that the b-EhCAl polypeptide was posttranslationally modified and processed into\ud two distinct proteins. In addition, preliminary data suggests that the mRNA may be\ud differentially processed in calcifying versus noncalcifying cells. Real time RT-PCR\ud experiments under bicarbonate limitation and over a 24 h time course suggest that\ud b-EhCAl performs different functions than the gamma and alpha CA isoforms\ud recently identified in E. huxleyi