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. 1994 Jul 19;91(15):6909–6913. doi: 10.1073/pnas.91.15.6909

A carbonic anhydrase from the archaeon Methanosarcina thermophila.

B E Alber 1, J G Ferry 1
PMCID: PMC44307  PMID: 8041719

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abbanat D. R., Ferry J. G. Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila. J Bacteriol. 1990 Dec;172(12):7145–7150. doi: 10.1128/jb.172.12.7145-7150.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Atkins C. A., Patterson B. D., Graham D. Plant Carbonic Anhydrases: II. Preparation and Some Properties of Monocotyledon and Dicotyledon Enzyme Types. Plant Physiol. 1972 Aug;50(2):218–223. doi: 10.1104/pp.50.2.218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bott M., Thauer R. K. The active species of "CO2" formed by carbon monoxide dehydrogenase from Peptostreptococcus productus. Z Naturforsch C. 1989 May-Jun;44(5-6):392–396. doi: 10.1515/znc-1989-5-609. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Bröckl G., Behr M., Fabry S., Hensel R., Kaudewitz H., Biendl E., König H. Analysis and nucleotide sequence of the genes encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis. Eur J Biochem. 1991 Jul 1;199(1):147–152. doi: 10.1111/j.1432-1033.1991.tb16102.x. [DOI] [PubMed] [Google Scholar]
  7. Clements A. P., Ferry J. G. Cloning, nucleotide sequence, and transcriptional analyses of the gene encoding a ferredoxin from Methanosarcina thermophila. J Bacteriol. 1992 Aug;174(16):5244–5250. doi: 10.1128/jb.174.16.5244-5250.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dharmavaram R., Gillevet P., Konisky J. Nucleotide sequence of the gene encoding the vanadate-sensitive membrane-associated ATPase of Methanococcus voltae. J Bacteriol. 1991 Mar;173(6):2131–2133. doi: 10.1128/jb.173.6.2131-2133.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dunn R., McCoy J., Simsek M., Majumdar A., Chang S. H., Rajbhandary U. L., Khorana H. G. The bacteriorhodopsin gene. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6744–6748. doi: 10.1073/pnas.78.11.6744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fawcett T. W., Browse J. A., Volokita M., Bartlett S. G. Spinach carbonic anhydrase primary structure deduced from the sequence of a cDNA clone. J Biol Chem. 1990 Apr 5;265(10):5414–5417. [PubMed] [Google Scholar]
  12. Ferry J. G. Methane from acetate. J Bacteriol. 1992 Sep;174(17):5489–5495. doi: 10.1128/jb.174.17.5489-5495.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fukuzawa H., Fujiwara S., Yamamoto Y., Dionisio-Sese M. L., Miyachi S. cDNA cloning, sequence, and expression of carbonic anhydrase in Chlamydomonas reinhardtii: regulation by environmental CO2 concentration. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4383–4387. doi: 10.1073/pnas.87.11.4383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fukuzawa H., Suzuki E., Komukai Y., Miyachi S. A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4437–4441. doi: 10.1073/pnas.89.10.4437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hausner W., Frey G., Thomm M. Control regions of an archaeal gene. A TATA box and an initiator element promote cell-free transcription of the tRNA(Val) gene of Methanococcus vannielii. J Mol Biol. 1991 Dec 5;222(3):495–508. doi: 10.1016/0022-2836(91)90492-o. [DOI] [PubMed] [Google Scholar]
  16. Jablonski P. E., DiMarco A. A., Bobik T. A., Cabell M. C., Ferry J. G. Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila. J Bacteriol. 1990 Mar;172(3):1271–1275. doi: 10.1128/jb.172.3.1271-1275.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kalmokoff M. L., Jarrell K. F. Cloning and sequencing of a multigene family encoding the flagellins of Methanococcus voltae. J Bacteriol. 1991 Nov;173(22):7113–7125. doi: 10.1128/jb.173.22.7113-7125.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krebs J. F., Fierke C. A., Alexander R. S., Christianson D. W. Conformational mobility of His-64 in the Thr-200----Ser mutant of human carbonic anhydrase II. Biochemistry. 1991 Sep 24;30(38):9153–9160. doi: 10.1021/bi00102a005. [DOI] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lechner J., Sumper M. The primary structure of a procaryotic glycoprotein. Cloning and sequencing of the cell surface glycoprotein gene of halobacteria. J Biol Chem. 1987 Jul 15;262(20):9724–9729. [PubMed] [Google Scholar]
  21. Lin X., Tang J. Purification, characterization, and gene cloning of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius. J Biol Chem. 1990 Jan 25;265(3):1490–1495. [PubMed] [Google Scholar]
  22. Maren T. H., Couto E. O. The nature of anion inhibition of human red cell carbonic anhydrases. Arch Biochem Biophys. 1979 Sep;196(2):501–510. doi: 10.1016/0003-9861(79)90302-3. [DOI] [PubMed] [Google Scholar]
  23. Nagao Y., Platero J. S., Waheed A., Sly W. S. Human mitochondrial carbonic anhydrase: cDNA cloning, expression, subcellular localization, and mapping to chromosome 16. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7623–7627. doi: 10.1073/pnas.90.16.7623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Okuyama T., Sato S., Zhu X. L., Waheed A., Sly W. S. Human carbonic anhydrase IV: cDNA cloning, sequence comparison, and expression in COS cell membranes. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1315–1319. doi: 10.1073/pnas.89.4.1315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ponticello G. S., Freedman M. B., Habecker C. N., Lyle P. A., Schwam H., Varga S. L., Christy M. E., Randall W. C., Baldwin J. J. Thienothiopyran-2-sulfonamides: a novel class of water-soluble carbonic anhydrase inhibitors. J Med Chem. 1987 Apr;30(4):591–597. doi: 10.1021/jm00387a002. [DOI] [PubMed] [Google Scholar]
  26. Price G. D., Howitt S. M., Harrison K., Badger M. R. Analysis of a genomic DNA region from the cyanobacterium Synechococcus sp. strain PCC7942 involved in carboxysome assembly and function. J Bacteriol. 1993 May;175(10):2871–2879. doi: 10.1128/jb.175.10.2871-2879.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reeve J. N. Molecular biology of methanogens. Annu Rev Microbiol. 1992;46:165–191. doi: 10.1146/annurev.mi.46.100192.001121. [DOI] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sanyal G., Pessah N. I., Maren T. H. Kinetics and inhibition of membrane-bound carbonic anhydrase from canine renal cortex. Biochim Biophys Acta. 1981 Jan 15;657(1):128–137. doi: 10.1016/0005-2744(81)90136-4. [DOI] [PubMed] [Google Scholar]
  30. Sanyal G., Swenson E. R., Pessah N. I., Maren T. H. The carbon dioxide hydration activity of skeletal muscle carbonic anhydrase. Inhibition by sulfonamides and anions. Mol Pharmacol. 1982 Jul;22(1):211–220. [PubMed] [Google Scholar]
  31. Simonen M., Palva I. Protein secretion in Bacillus species. Microbiol Rev. 1993 Mar;57(1):109–137. doi: 10.1128/mr.57.1.109-137.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  33. Sumper M., Berg E., Mengele R., Strobel I. Primary structure and glycosylation of the S-layer protein of Haloferax volcanii. J Bacteriol. 1990 Dec;172(12):7111–7118. doi: 10.1128/jb.172.12.7111-7118.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sung Y. C., Fuchs J. A. Characterization of the cyn operon in Escherichia coli K12. J Biol Chem. 1988 Oct 15;263(29):14769–14775. [PubMed] [Google Scholar]
  35. Tashian R. E. The carbonic anhydrases: widening perspectives on their evolution, expression and function. Bioessays. 1989 Jun;10(6):186–192. doi: 10.1002/bies.950100603. [DOI] [PubMed] [Google Scholar]
  36. Thauer R. K., Möller-Zinkhan D., Spormann A. M. Biochemistry of acetate catabolism in anaerobic chemotrophic bacteria. Annu Rev Microbiol. 1989;43:43–67. doi: 10.1146/annurev.mi.43.100189.000355. [DOI] [PubMed] [Google Scholar]
  37. Wade R., Gunning P., Eddy R., Shows T., Kedes L. Nucleotide sequence, tissue-specific expression, and chromosome location of human carbonic anhydrase III: the human CAIII gene is located on the same chromosome as the closely linked CAI and CAII genes. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9571–9575. doi: 10.1073/pnas.83.24.9571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Woese C. R., Kandler O., Wheelis M. L. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4576–4579. doi: 10.1073/pnas.87.12.4576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yao V. J., Spudich J. L. Primary structure of an archaebacterial transducer, a methyl-accepting protein associated with sensory rhodopsin I. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11915–11919. doi: 10.1073/pnas.89.24.11915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yu J. W., Price G. D., Song L., Badger M. R. Isolation of a Putative Carboxysomal Carbonic Anhydrase Gene from the Cyanobacterium Synechococcus PCC7942. Plant Physiol. 1992 Oct;100(2):794–800. doi: 10.1104/pp.100.2.794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

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