Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Apr;178(8):2368–2374. doi: 10.1128/jb.178.8.2368-2374.1996

Carboxyl-terminal processing of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus requires the hoxW gene product.

S Thiemermann 1, J Dernedde 1, M Bernhard 1, W Schroeder 1, C Massanz 1, B Friedrich 1
PMCID: PMC177947  PMID: 8636040

Abstract

Two open reading frames (ORFs) were identified immediately downstream of the four structural genes for the soluble hydrogenase (SH) of Alcaligenes eutrophus H16. While a mutation in ORF2 had no obvious effect on hydrogen metabolism, an in-frame deletion in ORF1, subsequently designated hoxW, led to a complete loss of SH activity and hence a significant retardation of autotrophic growth on hydrogen. Hydrogen oxidation in the hoxW mutant was catalyzed by the second hydrogenase, a membrane-bound enzyme. Assembly of the four subunits of the SH was blocked in mutant cells, and HoxH, the hydrogen-activating subunit, accumulated as a precursor which was still capable of binding nickel. Protein sequencing revealed that HoxH isolated from the wild type terminates at His-464, whereas the C-terminal amino acid sequence of HoxH from the hoxW mutant is colinear with the deduced sequence. Processing of the HoxH precursor was restored in vitro by a cell extract containing HoxW. These results indicate that HoxW is a highly specific carboxyl-terminal protease which releases a 24-amino-acid peptide from HoxH prior to progression of subunit assembly.

Full Text

The Full Text of this article is available as a PDF (421.1 KB).

Selected References

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

  1. 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]
  2. Anbudurai P. R., Mor T. S., Ohad I., Shestakov S. V., Pakrasi H. B. The ctpA gene encodes the C-terminal processing protease for the D1 protein of the photosystem II reaction center complex. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):8082–8086. doi: 10.1073/pnas.91.17.8082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bowien B., Schlegel H. G. Physiology and biochemistry of aerobic hydrogen-oxidizing bacteria. Annu Rev Microbiol. 1981;35:405–452. doi: 10.1146/annurev.mi.35.100181.002201. [DOI] [PubMed] [Google Scholar]
  4. Dernedde J., Eitinger M., Friedrich B. Analysis of a pleiotropic gene region involved in formation of catalytically active hydrogenases in Alcaligenes eutrophus H16. Arch Microbiol. 1993;159(6):545–553. doi: 10.1007/BF00249034. [DOI] [PubMed] [Google Scholar]
  5. Dernedde J., Eitinger T., Patenge N., Friedrich B. hyp gene products in Alcaligenes eutrophus are part of a hydrogenase-maturation system. Eur J Biochem. 1996 Jan 15;235(1-2):351–358. doi: 10.1111/j.1432-1033.1996.00351.x. [DOI] [PubMed] [Google Scholar]
  6. Friedrich B., Schwartz E. Molecular biology of hydrogen utilization in aerobic chemolithotrophs. Annu Rev Microbiol. 1993;47:351–383. doi: 10.1146/annurev.mi.47.100193.002031. [DOI] [PubMed] [Google Scholar]
  7. Gollin D. J., Mortenson L. E., Robson R. L. Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii. FEBS Lett. 1992 Sep 14;309(3):371–375. doi: 10.1016/0014-5793(92)80809-u. [DOI] [PubMed] [Google Scholar]
  8. Gough J. A., Murray N. E. Sequence diversity among related genes for recognition of specific targets in DNA molecules. J Mol Biol. 1983 May 5;166(1):1–19. doi: 10.1016/s0022-2836(83)80047-3. [DOI] [PubMed] [Google Scholar]
  9. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  10. Kortlüke C., Friedrich B. Maturation of membrane-bound hydrogenase of Alcaligenes eutrophus H16. J Bacteriol. 1992 Oct;174(19):6290–6293. doi: 10.1128/jb.174.19.6290-6293.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Lambin P., Fine J. M. Molecular weight estimation of proteins by electrophoresis in linear polyacrylamide gradient gels in the absence of denaturing agents. Anal Biochem. 1979 Sep 15;98(1):160–168. doi: 10.1016/0003-2697(79)90721-8. [DOI] [PubMed] [Google Scholar]
  14. Lenz O., Schwartz E., Dernedde J., Eitinger M., Friedrich B. The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation. J Bacteriol. 1994 Jul;176(14):4385–4393. doi: 10.1128/jb.176.14.4385-4393.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Mobley H. L., Island M. D., Hausinger R. P. Molecular biology of microbial ureases. Microbiol Rev. 1995 Sep;59(3):451–480. doi: 10.1128/mr.59.3.451-480.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pappin D. J., Findlay J. B. Sequence variability in the retinal-attachment domain of mammalian rhodopsins. Biochem J. 1984 Feb 1;217(3):605–613. doi: 10.1042/bj2170605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rossmann R., Maier T., Lottspeich F., Böck A. Characterisation of a protease from Escherichia coli involved in hydrogenase maturation. Eur J Biochem. 1995 Jan 15;227(1-2):545–550. doi: 10.1111/j.1432-1033.1995.tb20422.x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Sauter M., Böhm R., Böck A. Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli. Mol Microbiol. 1992 Jun;6(11):1523–1532. doi: 10.1111/j.1365-2958.1992.tb00873.x. [DOI] [PubMed] [Google Scholar]
  23. Schink B., Schlegel H. G. The membrane-bound hydrogenase of Alcaligenes eutrophus. I. Solubilization, purification, and biochemical properties. Biochim Biophys Acta. 1979 Apr 12;567(2):315–324. doi: 10.1016/0005-2744(79)90117-7. [DOI] [PubMed] [Google Scholar]
  24. Schmitz O., Boison G., Hilscher R., Hundeshagen B., Zimmer W., Lottspeich F., Bothe H. Molecular biological analysis of a bidirectional hydrogenase from cyanobacteria. Eur J Biochem. 1995 Oct 1;233(1):266–276. doi: 10.1111/j.1432-1033.1995.266_1.x. [DOI] [PubMed] [Google Scholar]
  25. Schneider K., Cammack R., Schlegel H. G. Content and localization of FMN, Fe-S clusters and nickel in the NAD-linked hydrogenase of Nocardia opaca 1b. Eur J Biochem. 1984 Jul 2;142(1):75–84. doi: 10.1111/j.1432-1033.1984.tb08252.x. [DOI] [PubMed] [Google Scholar]
  26. Schneider K., Pinkwart M., Jochim K. Purification of hydrogenases by affinity chromatography on Procion Red-agarose. Biochem J. 1983 Aug 1;213(2):391–398. doi: 10.1042/bj2130391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schneider K., Schlegel H. G. Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16. Biochim Biophys Acta. 1976 Nov 8;452(1):66–80. doi: 10.1016/0005-2744(76)90058-9. [DOI] [PubMed] [Google Scholar]
  28. Sorgenfrei O., Linder D., Karas M., Klein A. A novel very small subunit of a selenium containing [NiFe] hydrogenase of Methanococcus voltae is postranslationally processed by cleavage at a defined position. Eur J Biochem. 1993 May 1;213(3):1355–1358. doi: 10.1111/j.1432-1033.1993.tb17888.x. [DOI] [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. Tran-Betcke A., Warnecke U., Böcker C., Zaborosch C., Friedrich B. Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16. J Bacteriol. 1990 Jun;172(6):2920–2929. doi: 10.1128/jb.172.6.2920-2929.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Walker J. E. The NADH:ubiquinone oxidoreductase (complex I) of respiratory chains. Q Rev Biophys. 1992 Aug;25(3):253–324. doi: 10.1017/s003358350000425x. [DOI] [PubMed] [Google Scholar]
  33. Weidner U., Geier S., Ptock A., Friedrich T., Leif H., Weiss H. The gene locus of the proton-translocating NADH: ubiquinone oxidoreductase in Escherichia coli. Organization of the 14 genes and relationship between the derived proteins and subunits of mitochondrial complex I. J Mol Biol. 1993 Sep 5;233(1):109–122. doi: 10.1006/jmbi.1993.1488. [DOI] [PubMed] [Google Scholar]

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

RESOURCES