Amino Acid Sequence Homology between N- and C-terminal Halves of a Carbonic Anhydrase in Porphyridium purpureum, as Deduced from the Cloned cDNA

Abstract

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, comprising 1,960 base pairs, contained an open reading frame which encoded a 571-amino acid polypeptide with a calculated molecular mass of 62,094 Da.The N-and C-terminal halves of the putative mature Porphyridium CA have amino acid sequence homology to each other (>70%) and to other prokaryotic-type CAs. Both regions contain, at equivalent positions, one set of three possible zinc-liganding amino acid residues conserved among prokaryotic-type CAs. CA purified from Porphyridium contained two atoms of zinc per molecule. We propose that the Porphyridium CA has evolved by duplication of an ancestral CA gene followed by the fusion of the duplicated CA gene. The CA truncated into the putative mature form was overexpressed in Escherichia coli, and the expressed protein was active. Clones expressing separately the N-and C-terminal halves of the CA were constructed. CA activity was present in extracts of E. coli cells expressing the N-terminal half, while no detectable activity was found in cells expressing the C-terminal half.

Carbonic Anhydrase Is Essential for Growth of Ralstonia eutropha at Ambient CO 2 Concentrations

“…The amino acid sequence of the Can protein is more than 50% identical to those of putative ␤-CAs identified in Xylella fastidiosa (49) and P. aeruginosa (55), the almost-identical PCA1 and PCA2 of the red alga Porphyridium purpureum (41), and the yadF-encoded CA of E. coli (13). However, ␤-CAs are highly diverse, with overall sequence identities ranging to below 25%, limiting the similarities almost to the Zn 2ϩ -binding motifs within the catalytic centers of the enzymes (Fig 2).…”

Characterization and expression of the maize β-carbonic anhydrase gene repeat regions

“…The unicellular red alga, Porphyridium purpureum has a 62 kDa b-CA that appears to be a product of gene duplication, with the N-and C-terminal halves of the mature enzyme having sequence homology [22]. This arrangement has also been observed in the a-CA gene family, with a 63 kDa enzyme from Dunaliella salina shown to have two active sites [23], and a dual-domain CA has been identified in the giant clam Tridacna gigas.…”

“…However, in this case the C-and N-terminal CA domains have little amino acid homology (only 29% homology) [20]. It has been suggested that expression of the multiple domain protein results in a similar arrangement that arises from the association of monomer subunits of other b-CAs to produce active enzyme [22].…”

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

“…[3,4] In addition, carbonic anhydrases have been obtained and characterised from plant, yeast and bacteria. [4][5][6][7][8][9][10] The important roles of the enzyme in various cell types have been extensively reviewed. [4,5] At present, 14 isozymes (I-XIV) have been reported and found to be distributed throughout living organisms.…”