CLONING, CHARACTERIZATION AND EXPRESSION OF CARBONIC-ANHYDRASE FROM THE CYANOBACTERIUM SYNECHOCYSTIS PCC6803

A 3.3 kb HindIII restriction-digest DNA fragment was isolated from a S ynechocystis sp. strain PCC6803 subgenomic plasmid library which stron gly hybridized to a 349 bp fragment of the icfA (ccaA) gene from Synec hococcus sp. attain PCC7942. DNA sequence analysis of the fragment rev ealed three open reading frames (ORFs), two of which potentially coded for pantothenate synthetase (ORF275) and cytidylate kinase (ORF230). The third, ORF274, was 825 bp in length, encoding a deduced polypeptid e of 274 aa (M-r, 30747) that bears 55% sequence identity to the Synec hococcus icfA (ccaA) translation product, a beta-type carbonic anhydra se (CA). A 932 bp EcoRI fragment containing ORF274 was subcloned into an expression vector and the construct was transformed into Escherichi a coli for overexpression. Electrometric assays for CA activity reveal ed that whole cell extracts containing the recombinant protein signifi cantly enhanced the rate of conversion of CO2 to HCO3- and that 98% of this catalytic activity was inhibited by ethoxyzolamide, a well-chara cterized CA inhibitor. Antisera derived against the overexpressed prot ein recognized a 30.7 kDa protein that was predominantly associated wi th the isolated carboxysome fraction from Synechocystis. These results provide molecular and physiological evidence for the identification o f a ccaA homologue in Synechocystis PCC6803 that encodes a carboxysoma l beta-type CA.