Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Oct;179(19):6053–6060. doi: 10.1128/jb.179.19.6053-6060.1997

Purification and molecular characterization of the H2 uptake membrane-bound NiFe-hydrogenase from the carboxidotrophic bacterium Oligotropha carboxidovorans.

B Santiago 1, O Meyer 1
PMCID: PMC179508  PMID: 9324252

Abstract

The membrane-bound hydrogenase of Oligotropha carboxidovorans was solubilized with n-dodecyl-beta-D-maltoside and purified 28-fold with a yield of 29% and a specific activity of 173 to 178 micromol of H2 x min(-1) x mg(-1). It is the first hydrogenase studied in a carboxidotrophic bacterium. The enzyme acts on artificial electron-accepting dyes, such as methylene blue, but is ineffective with pyridine nucleotides or other soluble physiological electron acceptors. Hydrogenase of O. carboxidovorans belongs to class I of hydrogenases and is a heterodimeric 101,692-Da NiFe-protein composed of the polypeptides HoxL and HoxS. Molecular cloning data revealed, that HoxL comprises 604 amino acid residues and has a molecular mass of 67,163 Da. Pre-HoxS comprises 360 amino acid residues and is synthesized as a precursor protein which is cleaved after alanine at position 45, thus producing a mature HoxS of 33,767 Da. The leader sequence corresponds to the signal peptide of small subunits of hydrogenases. The hydropathy plots of HoxL and HoxS were indicative for the absence of transmembranous helices. HoxZ has four transmembranous helices and is considered the potential membrane anchor of hydrogenase in O. carboxidovorans. Hydrogenase genes show the transcriptional order 5' hoxV --> hoxS --> hoxL --> hoxZ 3'. The hox gene cluster as well as the clustered CO dehydrogenase (cox) and Calvin cycle (cbb) genes are arranged within a 30-kb DNA segment of the 128-kb megaplasmid pHCG3 of O. carboxidovorans.

Full Text

The Full Text of this article is available as a PDF (896.8 KB).

Selected References

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

  1. Adams M. W., Mortenson L. E., Chen J. S. Hydrogenase. Biochim Biophys Acta. 1980 Dec;594(2-3):105–176. doi: 10.1016/0304-4173(80)90007-5. [DOI] [PubMed] [Google Scholar]
  2. Black L. K., Fu C., Maier R. J. Sequences and characterization of hupU and hupV genes of Bradyrhizobium japonicum encoding a possible nickel-sensing complex involved in hydrogenase expression. J Bacteriol. 1994 Nov;176(22):7102–7106. doi: 10.1128/jb.176.22.7102-7106.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Cauvin B., Colbeau A., Vignais P. M. The hydrogenase structural operon in Rhodobacter capsulatus contains a third gene, hupM, necessary for the formation of a physiologically competent hydrogenase. Mol Microbiol. 1991 Oct;5(10):2519–2527. doi: 10.1111/j.1365-2958.1991.tb02098.x. [DOI] [PubMed] [Google Scholar]
  5. Dross F., Geisler V., Lenger R., Theis F., Krafft T., Fahrenholz F., Kojro E., Duchêne A., Tripier D., Juvenal K. The quinone-reactive Ni/Fe-hydrogenase of Wolinella succinogenes. Eur J Biochem. 1992 May 15;206(1):93–102. doi: 10.1111/j.1432-1033.1992.tb16905.x. [DOI] [PubMed] [Google Scholar]
  6. Elsen S., Colbeau A., Chabert J., Vignais P. M. The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus. J Bacteriol. 1996 Sep;178(17):5174–5181. doi: 10.1128/jb.178.17.5174-5181.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Fox J. D., He Y., Shelver D., Roberts G. P., Ludden P. W. Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum. J Bacteriol. 1996 Nov;178(21):6200–6208. doi: 10.1128/jb.178.21.6200-6208.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fox J. D., Kerby R. L., Roberts G. P., Ludden P. W. Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme. J Bacteriol. 1996 Mar;178(6):1515–1524. doi: 10.1128/jb.178.6.1515-1524.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goodrich J. A., Schwartz M. L., McClure W. R. Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res. 1990 Sep 11;18(17):4993–5000. doi: 10.1093/nar/18.17.4993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Hidalgo E., Leyva A., Ruiz-Argüeso T. Nucleotide sequence of the hydrogenase structural genes from Rhizobium leguminosarum. Plant Mol Biol. 1990 Aug;15(2):367–370. doi: 10.1007/BF00036924. [DOI] [PubMed] [Google Scholar]
  13. Kortlüke C., Horstmann K., Schwartz E., Rohde M., Binsack R., Friedrich B. A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16. J Bacteriol. 1992 Oct;174(19):6277–6289. doi: 10.1128/jb.174.19.6277-6289.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Menon A. L., Mortenson L. E., Robson R. L. Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii. J Bacteriol. 1992 Jul;174(14):4549–4557. doi: 10.1128/jb.174.14.4549-4557.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Menon A. L., Stults L. W., Robson R. L., Mortenson L. E. Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii. Gene. 1990 Nov 30;96(1):67–74. doi: 10.1016/0378-1119(90)90342-o. [DOI] [PubMed] [Google Scholar]
  18. Menon N. K., Robbins J., Der Vartanian M., Patil D., Peck H. D., Jr, Menon A. L., Robson R. L., Przybyla A. E. Carboxy-terminal processing of the large subunit of [NiFe] hydrogenases. FEBS Lett. 1993 Sep 27;331(1-2):91–95. doi: 10.1016/0014-5793(93)80303-c. [DOI] [PubMed] [Google Scholar]
  19. Meyer O., Schlegel H. G. Biology of aerobic carbon monoxide-oxidizing bacteria. Annu Rev Microbiol. 1983;37:277–310. doi: 10.1146/annurev.mi.37.100183.001425. [DOI] [PubMed] [Google Scholar]
  20. Meyer O., Schlegel H. G. Oxidation of carbon monoxide in cell extracts of Pseudomonas carboxydovorans. J Bacteriol. 1979 Feb;137(2):811–817. doi: 10.1128/jb.137.2.811-817.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Meyer O., Schlegel H. G. Reisolation of the carbon monoxide utilizing hydrogen bacterium Pseudomonas carboxydovorans (Kistner) comb. nov. Arch Microbiol. 1978 Jul;118(1):35–43. doi: 10.1007/BF00406071. [DOI] [PubMed] [Google Scholar]
  22. Nivière V., Wong S. L., Voordouw G. Site-directed mutagenesis of the hydrogenase signal peptide consensus box prevents export of a beta-lactamase fusion protein. J Gen Microbiol. 1992 Oct;138(10):2173–2183. doi: 10.1099/00221287-138-10-2173. [DOI] [PubMed] [Google Scholar]
  23. Pearson D. M., O'Reilly C., Colby J., Black G. W. DNA sequence of the cut A, B and C genes, encoding the molybdenum containing hydroxylase carbon monoxide dehydrogenase, from Pseudomonas thermocarboxydovorans strain C2. Biochim Biophys Acta. 1994 Dec 30;1188(3):432–438. doi: 10.1016/0005-2728(94)90066-3. [DOI] [PubMed] [Google Scholar]
  24. Rossmann R., Sauter M., Lottspeich F., Böck A. Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C-terminal processing at Arg537. Eur J Biochem. 1994 Mar 1;220(2):377–384. doi: 10.1111/j.1432-1033.1994.tb18634.x. [DOI] [PubMed] [Google Scholar]
  25. Schübel U., Kraut M., Mörsdorf G., Meyer O. Molecular characterization of the gene cluster coxMSL encoding the molybdenum-containing carbon monoxide dehydrogenase of Oligotropha carboxidovorans. J Bacteriol. 1995 Apr;177(8):2197–2203. doi: 10.1128/jb.177.8.2197-2203.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Vignais P. M., Toussaint B. Molecular biology of membrane-bound H2 uptake hydrogenases. Arch Microbiol. 1994;161(1):1–10. doi: 10.1007/BF00248887. [DOI] [PubMed] [Google Scholar]
  27. Volbeda A., Charon M. H., Piras C., Hatchikian E. C., Frey M., Fontecilla-Camps J. C. Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature. 1995 Feb 16;373(6515):580–587. doi: 10.1038/373580a0. [DOI] [PubMed] [Google Scholar]
  28. Wu L. F., Mandrand M. A. Microbial hydrogenases: primary structure, classification, signatures and phylogeny. FEMS Microbiol Rev. 1993 Apr;10(3-4):243–269. doi: 10.1111/j.1574-6968.1993.tb05870.x. [DOI] [PubMed] [Google Scholar]
  29. Yabuuchi E., Kosako Y., Yano I., Hotta H., Nishiuchi Y. Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. Nov.: Proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. Nov., Ralstonia solanacearum (Smith 1896) comb. Nov. and Ralstonia eutropha (Davis 1969) comb. Nov. Microbiol Immunol. 1995;39(11):897–904. doi: 10.1111/j.1348-0421.1995.tb03275.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES