A CARBONIC-ANHYDRASE FROM THE ARCHAEON METHANOSARCINA-THERMOPHILA

Carbonic anhydrase (CA) from acetate-grown Methanosarcina thermophila was purified >10,000-fold (22% recovery) to apparent homogeneity with a specific activity of 4872 units/mg. The estimated native molecular m ass of the enzyme is 84 kDa based on gel filtration chromatography. SD S/PAGE revealed one protein band with an apparent molecular mass of 40 kDa. The M. thermophila CA is less sensitive than human CA isozyme II toward inhibition by sulfonamides and monovalent ions. The gene encod ing this CA was cloned into pUC18 and sequenced. Escherichia coli harb oring the recombinant plasmid expresses CA activity (2.3 units/mg of c ell extract protein), Comparison of the deduced amino acid sequence wi th the N-terminal sequence of the purified protein shows that the gene encodes an additional 34 N-terminal residues with properties characte ristic of signal peptides in secretory proteins. The calculated molecu lar mass (22.9 kDa) and pI (4.0) suggest that SDS/PAGE overestimates t he submit size and that the native enzyme is a tetramer. To our knowle dge, the deduced amino acid sequence has no significant identity to an y known CA but has 35% sequence identity to the first 197 deduced N-te rminal amino acids of a proposed CO2-concentrating-mechanism protein f rom Synechococcus PCC7942 and 28% sequence identity to the deduced seq uence of ferripyochelin binding protein from Pseudomonas aeruginosa. T hus, our results indicate that this archaeal CA represents a distinct class of CAs and provide a basis to determine physiological roles for CA in acetotrophic anaerobes.