Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Jun;178(11):3270–3274. doi: 10.1128/jb.178.11.3270-3274.1996

Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila.

B E Alber 1, J G Ferry 1
PMCID: PMC178080  PMID: 8655508

Abstract

The gene encoding carbonic anhydrase from Methanosarcina thermophila was hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity. The enzyme purified from E. coli has properties (specific activity, inhibitor sensitivity, and thermostability) similar to those of the authentic enzyme isolated from M. thermophila; however, a discrepancy in molecular mass suggests that the carbonic anhydrase is posttranslationally modified in either E. coli or M. thermophila. Both the authentic and heterologously produced enzymes were stable to heating at 55 degrees C for 15 min but were inactivated at higher temperatures. No esterase activity was detected with p-nitrophenylacetate as the substrate. Plasma emission spectroscopy revealed approximately 0.6 Zn per subunit. As judged from the estimated native molecular mass, the enzyme is either a trimer or a tetramer. Western blot (immunoblot) analysis of cell extract proteins from M. thermophila indicates that the levels of carbonic anhydrase are regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells.

Full Text

The Full Text of this article is available as a PDF (373.8 KB).

Selected References

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

  1. Alber B. E., Ferry J. G. A carbonic anhydrase from the archaeon Methanosarcina thermophila. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6909–6913. doi: 10.1073/pnas.91.15.6909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong J. M., Myers D. V., Verpoorte J. A., Edsall J. T. Purification and properties of human erythrocyte carbonic anhydrases. J Biol Chem. 1966 Nov 10;241(21):5137–5149. [PubMed] [Google Scholar]
  3. Auld D. S. Metal-free dialysis tubing. Methods Enzymol. 1988;158:13–14. doi: 10.1016/0076-6879(88)58043-6. [DOI] [PubMed] [Google Scholar]
  4. Boriack-Sjodin P. A., Heck R. W., Laipis P. J., Silverman D. N., Christianson D. W. Structure determination of murine mitochondrial carbonic anhydrase V at 2.45-A resolution: implications for catalytic proton transfer and inhibitor design. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10949–10953. doi: 10.1073/pnas.92.24.10949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bracey M. H., Christiansen J., Tovar P., Cramer S. P., Bartlett S. G. Spinach carbonic anhydrase: investigation of the zinc-binding ligands by site-directed mutagenesis, elemental analysis, and EXAFS. Biochemistry. 1994 Nov 8;33(44):13126–13131. doi: 10.1021/bi00248a023. [DOI] [PubMed] [Google Scholar]
  6. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Eichler K., Bourgis F., Buchet A., Kleber H. P., Mandrand-Berthelot M. A. Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coli. Mol Microbiol. 1994 Sep;13(5):775–786. doi: 10.1111/j.1365-2958.1994.tb00470.x. [DOI] [PubMed] [Google Scholar]
  8. Eriksson A. E., Liljas A. Refined structure of bovine carbonic anhydrase III at 2.0 A resolution. Proteins. 1993 May;16(1):29–42. doi: 10.1002/prot.340160104. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Guilloton M. B., Korte J. J., Lamblin A. F., Fuchs J. A., Anderson P. M. Carbonic anhydrase in Escherichia coli. A product of the cyn operon. J Biol Chem. 1992 Feb 25;267(6):3731–3734. [PubMed] [Google Scholar]
  12. Holmquist B. Elimination of adventitious metals. Methods Enzymol. 1988;158:6–12. doi: 10.1016/0076-6879(88)58042-4. [DOI] [PubMed] [Google Scholar]
  13. Hunt J. B., Rhee M. J., Storm C. B. A rapid and convenient preparation of apocarbonic anhydrase. Anal Biochem. 1977 May 1;79(1-2):614–617. doi: 10.1016/0003-2697(77)90444-4. [DOI] [PubMed] [Google Scholar]
  14. Håkansson K., Carlsson M., Svensson L. A., Liljas A. Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes. J Mol Biol. 1992 Oct 20;227(4):1192–1204. doi: 10.1016/0022-2836(92)90531-n. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Kannan K. K., Ramanadham M., Jones T. A. Structure, refinement, and function of carbonic anhydrase isozymes: refinement of human carbonic anhydrase I. Ann N Y Acad Sci. 1984;429:49–60. doi: 10.1111/j.1749-6632.1984.tb12314.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Lechner J., Wieland F. Structure and biosynthesis of prokaryotic glycoproteins. Annu Rev Biochem. 1989;58:173–194. doi: 10.1146/annurev.bi.58.070189.001133. [DOI] [PubMed] [Google Scholar]
  19. Liljas A., Håkansson K., Jonsson B. H., Xue Y. Inhibition and catalysis of carbonic anhydrase. Recent crystallographic analyses. Eur J Biochem. 1994 Jan 15;219(1-2):1–10. doi: 10.1007/978-3-642-79502-2_1. [DOI] [PubMed] [Google Scholar]
  20. Lovley D. R., Ferry J. G. Production and Consumption of H(2) during Growth of Methanosarcina spp. on Acetate. Appl Environ Microbiol. 1985 Jan;49(1):247–249. doi: 10.1128/aem.49.1.247-249.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pocker Y., Sarkanen S. Carbonic anhydrase: structure catalytic versatility, and inhibition. Adv Enzymol Relat Areas Mol Biol. 1978;47:149–274. doi: 10.1002/9780470122921.ch3. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Rowlett R. S., Chance M. R., Wirt M. D., Sidelinger D. E., Royal J. R., Woodroffe M., Wang Y. F., Saha R. P., Lam M. G. Kinetic and structural characterization of spinach carbonic anhydrase. Biochemistry. 1994 Nov 29;33(47):13967–13976. doi: 10.1021/bi00251a003. [DOI] [PubMed] [Google Scholar]
  25. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  26. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Tobin A. J. Carbonic anhydrase from parsley leaves. J Biol Chem. 1970 May 25;245(10):2656–2666. [PubMed] [Google Scholar]
  29. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES