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. 1993 Sep;175(18):5867–5876. doi: 10.1128/jb.175.18.5867-5876.1993

Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16.

R A Siddiqui 1, U Warnecke-Eberz 1, A Hengsberger 1, B Schneider 1, S Kostka 1, B Friedrich 1
PMCID: PMC206666  PMID: 8376334

Abstract

Alcaligenes eutrophus H16 shows three distinct nitrate reductase activities (U. Warnecke-Eberz and B. Friedrich, Arch. Microbiol. 159:405-409, 1993). The periplasmic enzyme, designated NAP (nitrate reductase, periplasmic), has been isolated. The 80-fold-purified heterodimeric enzyme catalyzed nitrate reduction with reduced viologen dyes as electron donors. The nap genes were identified in a library of A. eutrophus H16 megaplasmid DNA by using oligonucleotide probes based on the amino-terminal polypeptide sequences of the two NAP subunits. The two structural genes, designated napA and napB, code for polypeptides of 93 and 18.9 kDa, respectively. Sequence comparisons indicate that the putative gene products are translated with signal peptides of 28 and 35 amino acids, respectively. This is compatible with the fact that NAP activity was found in the soluble fraction of cell extracts and suggests that the mature enzyme is located in the periplasm. The deduced sequence of the large subunit, NAPA, contained two conserved amino-terminal stretches of amino acids found in molybdenum-dependent proteins such as nitrate reductases and formate dehydrogenases, suggesting that NAPA contains the catalytic site. The predicted sequence of the small subunit, NAPB, revealed two potential heme c-binding sites, indicating its involvement in the transfer of electrons. An insertion in the napA gene led to a complete loss of NAP activity but did not abolish the ability of A. eutrophus to use nitrate as a nitrogen source or as an electron acceptor in anaerobic respiration. Nevertheless, the NAP-deficient mutant showed delayed growth after transition from aerobic to anaerobic respiration, suggesting a role for NAP in the adaptation to anaerobic metabolism.

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Selected References

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

  1. Appleyard R K. Segregation of New Lysogenic Types during Growth of a Doubly Lysogenic Strain Derived from Escherichia Coli K12. Genetics. 1954 Jul;39(4):440–452. doi: 10.1093/genetics/39.4.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bell L. C., Richardson D. J., Ferguson S. J. Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha. The periplasmic enzyme catalyzes the first step in aerobic denitrification. FEBS Lett. 1990 Jun 4;265(1-2):85–87. doi: 10.1016/0014-5793(90)80889-q. [DOI] [PubMed] [Google Scholar]
  3. Berg B. L., Li J., Heider J., Stewart V. Nitrate-inducible formate dehydrogenase in Escherichia coli K-12. I. Nucleotide sequence of the fdnGHI operon and evidence that opal (UGA) encodes selenocysteine. J Biol Chem. 1991 Nov 25;266(33):22380–22385. [PubMed] [Google Scholar]
  4. Berg D. E., Davies J., Allet B., Rochaix J. D. Transposition of R factor genes to bacteriophage lambda. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3628–3632. doi: 10.1073/pnas.72.9.3628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blasco F., Iobbi C., Giordano G., Chippaux M., Bonnefoy V. Nitrate reductase of Escherichia coli: completion of the nucleotide sequence of the nar operon and reassessment of the role of the alpha and beta subunits in iron binding and electron transfer. Mol Gen Genet. 1989 Aug;218(2):249–256. doi: 10.1007/BF00331275. [DOI] [PubMed] [Google Scholar]
  6. Bokranz M., Gutmann M., Körtner C., Kojro E., Fahrenholz F., Lauterbach F., Kröger A. Cloning and nucleotide sequence of the structural genes encoding the formate dehydrogenase of Wolinella succinogenes. Arch Microbiol. 1991;156(2):119–128. doi: 10.1007/BF00290984. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Bruschi M., Guerlesquin F. Structure, function and evolution of bacterial ferredoxins. FEMS Microbiol Rev. 1988 Apr-Jun;4(2):155–175. doi: 10.1111/j.1574-6968.1988.tb02741.x. [DOI] [PubMed] [Google Scholar]
  9. Eberz G., Friedrich B. Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus. J Bacteriol. 1991 Mar;173(6):1845–1854. doi: 10.1128/jb.173.6.1845-1854.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eitinger T., Friedrich B. Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus. J Biol Chem. 1991 Feb 15;266(5):3222–3227. [PubMed] [Google Scholar]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. Hochstein L. I., Tomlinson G. A. The enzymes associated with denitrification. Annu Rev Microbiol. 1988;42:231–261. doi: 10.1146/annurev.mi.42.100188.001311. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. LOWE R. H., EVANS H. J. PREPARATION AND SOME PROPERTIES OF A SOLUBLE NITRATE REDUCTASE FROM RHIZOBIUM JAPONICUM. Biochim Biophys Acta. 1964 Jun 1;85:377–389. doi: 10.1016/0926-6569(64)90301-3. [DOI] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. 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]
  19. Lin J. T., Goldman B. S., Stewart V. Structures of genes nasA and nasB, encoding assimilatory nitrate and nitrite reductases in Klebsiella pneumoniae M5al. J Bacteriol. 1993 Apr;175(8):2370–2378. doi: 10.1128/jb.175.8.2370-2378.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pfitzner J., Schlegel H. G. Denitrifikation bei Hydrogenomonas eutropha Stamm H 16. Arch Mikrobiol. 1973;90(3):199–211. [PubMed] [Google Scholar]
  21. Pierson D. E., Campbell A. Cloning and nucleotide sequence of bisC, the structural gene for biotin sulfoxide reductase in Escherichia coli. J Bacteriol. 1990 Apr;172(4):2194–2198. doi: 10.1128/jb.172.4.2194-2198.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pollock W. B., Loutfi M., Bruschi M., Rapp-Giles B. J., Wall J. D., Voordouw G. Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough. J Bacteriol. 1991 Jan;173(1):220–228. doi: 10.1128/jb.173.1.220-228.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Richardson D. J., McEwan A. G., Page M. D., Jackson J. B., Ferguson S. J. The identification of cytochromes involved in the transfer of electrons to the periplasmic NO3- reductase of Rhodobacter capsulatus and resolution of a soluble NO3(-)-reductase--cytochrome-c552 redox complex. Eur J Biochem. 1990 Nov 26;194(1):263–270. doi: 10.1111/j.1432-1033.1990.tb19452.x. [DOI] [PubMed] [Google Scholar]
  24. Robertson L. A., Kuenen J. G. Combined heterotrophic nitrification and aerobic denitrification in Thiosphaera pantotropha and other bacteria. Antonie Van Leeuwenhoek. 1990 Apr;57(3):139–152. doi: 10.1007/BF00403948. [DOI] [PubMed] [Google Scholar]
  25. Römermann D., Friedrich B. Denitrification by Alcaligenes eutrophus is plasmid dependent. J Bacteriol. 1985 May;162(2):852–854. doi: 10.1128/jb.162.2.852-854.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Shuber A. P., Orr E. C., Recny M. A., Schendel P. F., May H. D., Schauer N. L., Ferry J. G. Cloning, expression, and nucleotide sequence of the formate dehydrogenase genes from Methanobacterium formicicum. J Biol Chem. 1986 Oct 5;261(28):12942–12947. [PubMed] [Google Scholar]
  28. Simon R., Quandt J., Klipp W. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Gene. 1989 Aug 1;80(1):161–169. doi: 10.1016/0378-1119(89)90262-x. [DOI] [PubMed] [Google Scholar]
  29. Stewart V. Nitrate respiration in relation to facultative metabolism in enterobacteria. Microbiol Rev. 1988 Jun;52(2):190–232. doi: 10.1128/mr.52.2.190-232.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stouthamer A. H. Biochemistry and genetics of nitrate reductase in bacteria. Adv Microb Physiol. 1976;14(11):315–375. doi: 10.1016/s0065-2911(08)60230-1. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Zinoni F., Birkmann A., Stadtman T. C., Böck A. Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4650–4654. doi: 10.1073/pnas.83.13.4650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. de Bruijn F. J., Lupski J. R. The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids--a review. Gene. 1984 Feb;27(2):131–149. doi: 10.1016/0378-1119(84)90135-5. [DOI] [PubMed] [Google Scholar]

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