Catalytic exchange of 18O from 13C18O-labelled CO2 by wild-type cells and ecaA, ecaB, and ccaA mutants of the cyanobacteria Synechococcus PCC7942 and Synechocystis PCC6803

Expression constructs carrying the ecaA, ecaB, and ccaA genes from the cyanobacteria Synechococcus PCC7942 and Synechocystis PCC6803 were generated and each transformed into Escherichia coli. Lysates from cells expressing recombinant protein were prepared and assayed using mass spectrometry for their ability to catalyze the exchange of O-18 from (CO2)-C-13-O-18 to H2O. No carbonic anhydrase (CA) activity was detected from the cell lysates containing recombinant EcaA or EcaB proteins, whereas the lysate harbouring the CcaA polypeptide clearly accelerated the rate of O-18 exchange. An ecaA deletion mutant in Synechococcus and insertionally inactivated ecaB and ccaA mutants in Synechocystis were generated and similarly assessed for CA activity. All mutants displayed a transient, ethoxyzolamide-sensitive, CA-like catalysis that was also exhibited by wild-type cells. The CcaA-deficient mutant showed a reduced capacity to exchange O-18 out of (CO)-C-13-O-18-labelled CO2 in the light as well as an absolute requirement for high C-i for growth, reflecting the importance of the carboxysomal CA to the operation of the cyanobacterial CO2-concentrating mechanism. No detectable differences in O-18 exchange patterns, CO2 or HCO3- transport, or steady-state growth were observed between the ecaA and ecaB mutants and wild-type cells, indicating that neither EcaA nor EcaB play an essential catalytic role in the functioning of the CO2-concentrating mechanism.