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 mass of the enzyme is 84 kDa based on gel filtration chromatography. SDS/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 encoding this CA was cloned into pUC18 and sequenced. Escherichia coli harboring the recombinant plasmid expresses CA activity (2.3 units/mg of cell extract protein). Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein shows that the gene encodes an additional 34 N-terminal residues with properties characteristic of signal peptides in secretory proteins. The calculated molecular mass (22.9 kDa) and pI (4.0) suggest that SDS/PAGE overestimates the subunit size and that the native enzyme is a tetramer. To our knowledge, the deduced amino acid sequence has no significant identity to any known CA but has 35% sequence identity to the first 197 deduced N-terminal amino acids of a proposed CO2-concentrating-mechanism protein from Synechococcus PCC7942 and 28% sequence identity to the deduced sequence of ferripyochelin binding protein from Pseudomonas aeruginosa. Thus, 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.
Alber
B E
BE
Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061-0305.
Ferry
J G
JG
D000595:Amino Acid Sequence; D001483:Base Sequence; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D017020:Methanosarcina / Q000201:enzymology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D002257:Carbonic Anhydrase Inhibitors; D004269:DNA, Bacterial; D002256:Carbonic Anhydrases