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. 1995 Jun;177(11):3111–3119. doi: 10.1128/jb.177.11.3111-3119.1995

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

E R Rocha 1, C J Smith 1
PMCID: PMC177000  PMID: 7768808

Abstract

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.

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Selected References

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  1. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–126. doi: 10.1016/s0076-6879(84)05016-3. [DOI] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bishai W. R., Smith H. O., Barcak G. J. A peroxide/ascorbate-inducible catalase from Haemophilus influenzae is homologous to the Escherichia coli katE gene product. J Bacteriol. 1994 May;176(10):2914–2921. doi: 10.1128/jb.176.10.2914-2921.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bol D. K., Yasbin R. E. The isolation, cloning and identification of a vegetative catalase gene from Bacillus subtilis. Gene. 1991 Dec 20;109(1):31–37. doi: 10.1016/0378-1119(91)90585-y. [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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Brown-Peterson N. J., Salin M. L. Purification of a catalase-peroxidase from Halobacterium halobium: characterization of some unique properties of the halophilic enzyme. J Bacteriol. 1993 Jul;175(13):4197–4202. doi: 10.1128/jb.175.13.4197-4202.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Claiborne A., Fridovich I. Purification of the o-dianisidine peroxidase from Escherichia coli B. Physicochemical characterization and analysis of its dual catalatic and peroxidatic activities. J Biol Chem. 1979 May 25;254(10):4245–4252. [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. Diaz G. A., Wayne L. G. Isolation and characterization of catalase produced by Mycobacterium tuberculosis. Am Rev Respir Dis. 1974 Sep;110(3):312–319. doi: 10.1164/arrd.1974.110.3.312. [DOI] [PubMed] [Google Scholar]
  10. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Farr S. B., Kogoma T. Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol Rev. 1991 Dec;55(4):561–585. doi: 10.1128/mr.55.4.561-585.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fita I., Rossmann M. G. The NADPH binding site on beef liver catalase. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1604–1608. doi: 10.1073/pnas.82.6.1604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fita I., Rossmann M. G. The active center of catalase. J Mol Biol. 1985 Sep 5;185(1):21–37. doi: 10.1016/0022-2836(85)90180-9. [DOI] [PubMed] [Google Scholar]
  14. Forkl H., Vandekerckhove J., Drews G., Tadros M. H. Molecular cloning, sequence analysis and expression of the gene for catalase-peroxidase (cpeA) from the photosynthetic bacterium Rhodobacter capsulatus B10. Eur J Biochem. 1993 May 15;214(1):251–258. doi: 10.1111/j.1432-1033.1993.tb17918.x. [DOI] [PubMed] [Google Scholar]
  15. Fridovich I. The biology of oxygen radicals. Science. 1978 Sep 8;201(4359):875–880. doi: 10.1126/science.210504. [DOI] [PubMed] [Google Scholar]
  16. Goldberg I., Hochman A. Purification and characterization of a novel type of catalase from the bacterium Klebsiella pneumoniae. Biochim Biophys Acta. 1989 May 31;991(2):330–336. doi: 10.1016/0304-4165(89)90124-4. [DOI] [PubMed] [Google Scholar]
  17. Gregory E. M., Dapper C. H. Isolation of iron-containing superoxide dismutase from Bacteroides fragilis: reconstitution as a Mn-containing enzyme. Arch Biochem Biophys. 1983 Jan;220(1):293–300. doi: 10.1016/0003-9861(83)90413-7. [DOI] [PubMed] [Google Scholar]
  18. Gregory E. M., Fanning D. D. Effect of heme on Bacteroides distasonis catalase and aerotolerance. J Bacteriol. 1983 Dec;156(3):1012–1018. doi: 10.1128/jb.156.3.1012-1018.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gregory E. M., Fridovich I. Visualization of catalase on acrylamide gels. Anal Biochem. 1974 Mar;58(1):57–62. doi: 10.1016/0003-2697(74)90440-0. [DOI] [PubMed] [Google Scholar]
  20. Gregory E. M., Kowalski J. B., Holdeman L. V. Production and some properties of catalase and superoxide dismutase from the anaerobe Bacteroides distasonis. J Bacteriol. 1977 Mar;129(3):1298–1302. doi: 10.1128/jb.129.3.1298-1302.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gregory E. M., Veltri B. J., Wagner D. L., Wilkins T. D. Carbohydrate repression of catalase synthesis in Bacteroides fragilis. J Bacteriol. 1977 Jan;129(1):534–535. doi: 10.1128/jb.129.1.534-535.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Haas A., Brehm K., Kreft J., Goebel W. Cloning, characterization, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases. J Bacteriol. 1991 Aug;173(16):5159–5167. doi: 10.1128/jb.173.16.5159-5167.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Halliwell B., Gutteridge J. M. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J. 1984 Apr 1;219(1):1–14. doi: 10.1042/bj2190001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hassan H. M., Fridovich I. Regulation of the synthesis of catalase and peroxidase in Escherichia coli. J Biol Chem. 1978 Sep 25;253(18):6445–6420. [PubMed] [Google Scholar]
  25. Hedrick J. L., Smith A. J. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys. 1968 Jul;126(1):155–164. doi: 10.1016/0003-9861(68)90569-9. [DOI] [PubMed] [Google Scholar]
  26. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  27. Imlay J. A., Linn S. DNA damage and oxygen radical toxicity. Science. 1988 Jun 3;240(4857):1302–1309. doi: 10.1126/science.3287616. [DOI] [PubMed] [Google Scholar]
  28. Knauf H. J., Vogel R. F., Hammes W. P. Cloning, sequence, and phenotypic expression of katA, which encodes the catalase of Lactobacillus sake LTH677. Appl Environ Microbiol. 1992 Mar;58(3):832–839. doi: 10.1128/aem.58.3.832-839.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Knoch M., van Pée K. H., Vining L. C., Lingens F. Purification, properties and immunological detection of a bromoperoxidase-catalase from Streptomyces venezuelae and from a chloramphenicol-nonproducing mutant. J Gen Microbiol. 1989 Sep;135(9):2493–2502. doi: 10.1099/00221287-135-9-2493. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Lange R., Hengge-Aronis R. Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol Microbiol. 1991 Jan;5(1):49–59. doi: 10.1111/j.1365-2958.1991.tb01825.x. [DOI] [PubMed] [Google Scholar]
  32. Loewen P. C., Stauffer G. V. Nucleotide sequence of katG of Salmonella typhimurium LT2 and characterization of its product, hydroperoxidase I. Mol Gen Genet. 1990 Oct;224(1):147–151. doi: 10.1007/BF00259461. [DOI] [PubMed] [Google Scholar]
  33. Loewen P. C., Switala J., Triggs-Raine B. L. Catalases HPI and HPII in Escherichia coli are induced independently. Arch Biochem Biophys. 1985 Nov 15;243(1):144–149. doi: 10.1016/0003-9861(85)90782-9. [DOI] [PubMed] [Google Scholar]
  34. Loewen P. C., Triggs B. L. Genetic mapping of katF, a locus that with katE affects the synthesis of a second catalase species in Escherichia coli. J Bacteriol. 1984 Nov;160(2):668–675. doi: 10.1128/jb.160.2.668-675.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Loprasert S., Negoro S., Okada H. Cloning, nucleotide sequence, and expression in Escherichia coli of the Bacillus stearothermophilus peroxidase gene (perA). J Bacteriol. 1989 Sep;171(9):4871–4875. doi: 10.1128/jb.171.9.4871-4875.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Loprasert S., Negoro S., Okada H. Thermostable peroxidase from Bacillus stearothermophilus. J Gen Microbiol. 1988 Jul;134(7):1971–1976. doi: 10.1099/00221287-134-7-1971. [DOI] [PubMed] [Google Scholar]
  37. Morris S. L., Nair J., Rouse D. A. The catalase-peroxidase of Mycobacterium intracellulare: nucleotide sequence analysis and expression in Escherichia coli. J Gen Microbiol. 1992 Nov;138(11):2363–2370. doi: 10.1099/00221287-138-11-2363. [DOI] [PubMed] [Google Scholar]
  38. Mulvey M. R., Sorby P. A., Triggs-Raine B. L., Loewen P. C. Cloning and physical characterization of katE and katF required for catalase HPII expression in Escherichia coli. Gene. 1988 Dec 20;73(2):337–345. doi: 10.1016/0378-1119(88)90498-2. [DOI] [PubMed] [Google Scholar]
  39. Murshudov G. N., Melik-Adamyan W. R., Grebenko A. I., Barynin V. V., Vagin A. A., Vainshtein B. K., Dauter Z., Wilson K. S. Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 A resolution. FEBS Lett. 1992 Nov 9;312(2-3):127–131. doi: 10.1016/0014-5793(92)80919-8. [DOI] [PubMed] [Google Scholar]
  40. Murthy M. R., Reid T. J., 3rd, Sicignano A., Tanaka N., Rossmann M. G. Structure of beef liver catalase. J Mol Biol. 1981 Oct 25;152(2):465–499. doi: 10.1016/0022-2836(81)90254-0. [DOI] [PubMed] [Google Scholar]
  41. Nelson D. P., Kiesow L. A. Enthalpy of decomposition of hydrogen peroxide by catalase at 25 degrees C (with molar extinction coefficients of H 2 O 2 solutions in the UV). Anal Biochem. 1972 Oct;49(2):474–478. doi: 10.1016/0003-2697(72)90451-4. [DOI] [PubMed] [Google Scholar]
  42. Onderdonk A. B., Johnston J., Mayhew J. W., Gorbach S. L. Effect of dissolved oxygen and Eh and Bacteroides fragilis during continuous culture. Appl Environ Microbiol. 1976 Feb;31(2):168–172. doi: 10.1128/aem.31.2.168-172.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Privitera G., Dublanchet A., Sebald M. Transfer of multiple antibiotic resistance between subspecies of Bacteroides fragilis. J Infect Dis. 1979 Jan;139(1):97–101. doi: 10.1093/infdis/139.1.97. [DOI] [PubMed] [Google Scholar]
  44. Putney S. D., Benkovic S. J., Schimmel P. R. A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7350–7354. doi: 10.1073/pnas.78.12.7350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rolfe R. D., Hentges D. J., Barrett J. T., Campbell B. J. Oxygen tolerance of human intestinal anaerobes. Am J Clin Nutr. 1977 Nov;30(11):1762–1769. doi: 10.1093/ajcn/30.11.1762. [DOI] [PubMed] [Google Scholar]
  46. Ryan T. P., Aust S. D. The role of iron in oxygen-mediated toxicities. Crit Rev Toxicol. 1992;22(2):119–141. doi: 10.3109/10408449209146308. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Schroeder W. A., Shelton J. R., Shelton J. B., Robberson B., Apell G., Fang R. S., Bonaventura J. The complete amino acid sequence of bovine liver catalase and the partial sequence of bovine erythrocyte catalase. Arch Biochem Biophys. 1982 Mar;214(1):397–421. doi: 10.1016/0003-9861(82)90044-3. [DOI] [PubMed] [Google Scholar]
  49. Shimoni M., Reuveni R. A method for staining and stabilizing peroxidase activity in polyacrylamide gel electrophoresis. Anal Biochem. 1988 Nov 15;175(1):35–38. doi: 10.1016/0003-2697(88)90357-0. [DOI] [PubMed] [Google Scholar]
  50. Sichak S. P., Dounce A. L. Analysis of the peroxidatic mode of action of catalase. Arch Biochem Biophys. 1986 Sep;249(2):286–295. doi: 10.1016/0003-9861(86)90004-4. [DOI] [PubMed] [Google Scholar]
  51. Smith C. J. Characterization of Bacteroides ovatus plasmid pBI136 and structure of its clindamycin resistance region. J Bacteriol. 1985 Mar;161(3):1069–1073. doi: 10.1128/jb.161.3.1069-1073.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Smith C. J., Owen C., Kirby L. Activation of a cryptic streptomycin-resistance gene in the Bacteroides erm transposon, Tn4551. Mol Microbiol. 1992 Aug;6(16):2287–2297. doi: 10.1111/j.1365-2958.1992.tb01404.x. [DOI] [PubMed] [Google Scholar]
  53. Southern J. A., Parker J. R., Woods D. R. Expression and purification of glutamine synthetase cloned from Bacteroides fragilis. J Gen Microbiol. 1986 Oct;132(10):2827–2835. doi: 10.1099/00221287-132-10-2827. [DOI] [PubMed] [Google Scholar]
  54. Storz G., Tartaglia L. A., Farr S. B., Ames B. N. Bacterial defenses against oxidative stress. Trends Genet. 1990 Nov;6(11):363–368. doi: 10.1016/0168-9525(90)90278-e. [DOI] [PubMed] [Google Scholar]
  55. Tally F. P., Stewart P. R., Sutter V. L., Rosenblatt J. E. Oxygen tolerance of fresh clinical anaerobic bacteria. J Clin Microbiol. 1975 Feb;1(2):161–164. doi: 10.1128/jcm.1.2.161-164.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Triggs-Raine B. L., Doble B. W., Mulvey M. R., Sorby P. A., Loewen P. C. Nucleotide sequence of katG, encoding catalase HPI of Escherichia coli. J Bacteriol. 1988 Sep;170(9):4415–4419. doi: 10.1128/jb.170.9.4415-4419.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Vainshtein B. K., Melik-Adamyan W. R., Barynin V. V., Vagin A. A., Grebenko A. I., Borisov V. V., Bartels K. S., Fita I., Rossmann M. G. Three-dimensional structure of catalase from Penicillium vitale at 2.0 A resolution. J Mol Biol. 1986 Mar 5;188(1):49–61. doi: 10.1016/0022-2836(86)90479-1. [DOI] [PubMed] [Google Scholar]
  58. Walden W. C., Hentges D. J. Differential effects of oxygen and oxidation-reduction potential on the multiplication of three species of anaerobic intestinal bacteria. Appl Microbiol. 1975 Nov;30(5):781–785. doi: 10.1128/am.30.5.781-785.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Weisburg W. G., Oyaizu Y., Oyaizu H., Woese C. R. Natural relationship between bacteroides and flavobacteria. J Bacteriol. 1985 Oct;164(1):230–236. doi: 10.1128/jb.164.1.230-236.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Welinder K. G. Bacterial catalase-peroxidases are gene duplicated members of the plant peroxidase superfamily. Biochim Biophys Acta. 1991 Nov 15;1080(3):215–220. doi: 10.1016/0167-4838(91)90004-j. [DOI] [PubMed] [Google Scholar]
  61. Wilkins T. D., Wagner D. L., Veltri B. J., Jr, Gregory E. M. Factors affecting production of catalase by Bacteroides. J Clin Microbiol. 1978 Nov;8(5):553–557. doi: 10.1128/jcm.8.5.553-557.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Woodbury W., Spencer A. K., Stahman M. A. An improved procedure using ferricyanide for detecting catalase isozymes. Anal Biochem. 1971 Nov;44(1):301–305. doi: 10.1016/0003-2697(71)90375-7. [DOI] [PubMed] [Google Scholar]
  63. Zhang Y., Heym B., Allen B., Young D., Cole S. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature. 1992 Aug 13;358(6387):591–593. doi: 10.1038/358591a0. [DOI] [PubMed] [Google Scholar]
  64. von Ossowski I., Mulvey M. R., Leco P. A., Borys A., Loewen P. C. Nucleotide sequence of Escherichia coli katE, which encodes catalase HPII. J Bacteriol. 1991 Jan;173(2):514–520. doi: 10.1128/jb.173.2.514-520.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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