AMINO-ACID-SEQUENCE HOMOLOGY BETWEEN N-TERMINAL AND C-TERMINAL HALVESOF A CARBONIC-ANHYDRASE IN PORPHYRIDIUM-PURPUREUM, AS DEDUCED FROM THE CLOTTED CDNA

Carbonic anhydrase (CA) from Porphyridium purpureum, a unicellular red alga, was purified >209-fold to a specific activity of 1,147 units/mg protein, cDNA clones for this CA were isolated. The longest clone, co mprising 1,960 base pairs, contained an open reading frame which encod ed a 571-amino acid polypeptide with a calculated molecular mass of 62 ,094 Da. The N- and C-terminal halves of the putative mature Porphyrid ium CA have amino acid sequence homology to each other (>70%) and to o ther prokaryotic-type CAs. Both regions contain, at equivalent positio ns, one set of three possible zinc-liganding amino acid residues conse rved among prokaryotic-type CAs. CA purified from Porphyridium contain ed two atoms of zinc per molecule. We propose that the Porphyridium CA has evolved by duplication of an ancestral CA gene followed by the fu sion of the duplicated CA gene. The CA truncated into the putative mat ure form was overexpressed in Escherichia coli, and the expressed prot ein was active. Clones expressing separately the N- and C-terminal hal ves of the CA were constructed. CA activity was present in extracts of E. coli cells expressing the N-terminal half, while no detectable act ivity was found in cells expressing the C-terminal half.