Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom Phaeodactylum tricornutum

A single intracellular carbonic anhydrase (CA) was detected in air-grown an d, at reduced levels, in high CO2-grown cells of the marine diatom Phaeodac tylum tricornutum (UTEX 642). No external CA activity was detected irrespec tive of growth CO2 conditions. Ethoxyzolamide (0.4 mm), a CA-specific inhib itor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mm acetazolamide had little effec t on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. t ricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on di ethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. Th e purified CA was shown, by SDS-PAGE, to comprise an electrophoretically si ngle polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplifica tion of cDNA ends method and indicated that the cDNA encodes 282 amino acid s. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible sig nal sequence of up to 46 amino acids at the N terminus. The mature CA was f ound to consist of 236 amino acids and the sequence was homologous to beta -type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO2-bi nding site appeared to be unique among the beta -CAs so far reported.