Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1995 May;108(1):203–210. doi: 10.1104/pp.108.1.203

The choice of reducing substrate is altered by replacement of an alanine by a proline in the FAD domain of a bispecific NAD(P)H-nitrate reductase from birch.

T Schöndorf 1, W Hachtel 1
PMCID: PMC157322  PMID: 7784504

Abstract

Differences in the amino acid sequence between the bispecific NAD(P)H-nitrate reductase of birch (Betula pendula Roth) and the monospecific NADH-nitrate reductases of a variety of other higher plants have been found at the dinucleotide-binding site in the FAD domain. To pinpoint amino acid residues that determine the choice of reducing substrate, we introduced mutations into the cDNA coding for birch nitrate reductase. These mutations were aimed at replacing certain amino acids of the NAD(P)H-binding site by conserved amino acids located at identical positions in NADH-monospecific enzymes. The mutated cDNAs were integrated into the genome of tobacco by Agrobacterium-mediated transformation. Transgenic tobacco (Nicotiana tabacum) plants were grown on a medium containing ammonium as the sole nitrogen source to keep endogenous tobacco nitrate reductase activity low. Whereas some of the mutated enzymes showed a slight preference for NADPH, as does the nonmutated birch enzyme, the activity of some others greatly depended on the availability of NADH and was low with NADPH alone. Comparison of the mutations reveals that replacement of a single amino acid in the birch sequence (alanine871 by proline) is critical for the use of reducing substrate.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Barber M. J., Solomonson L. P. The role of the essential sulfhydryl group in assimilatory NADH: nitrate reductase of Chlorella. J Biol Chem. 1986 Apr 5;261(10):4562–4567. [PubMed] [Google Scholar]
  2. Bevan M., Barnes W. M., Chilton M. D. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res. 1983 Jan 25;11(2):369–385. doi: 10.1093/nar/11.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bredt D. S., Hwang P. M., Glatt C. E., Lowenstein C., Reed R. R., Snyder S. H. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature. 1991 Jun 27;351(6329):714–718. doi: 10.1038/351714a0. [DOI] [PubMed] [Google Scholar]
  4. Cherel I., Grosclaude J., Rouze P. Monoclonal antibodies identify multiple epitopes on maize leaf nitrate reductase. Biochem Biophys Res Commun. 1985 Jun 28;129(3):686–693. doi: 10.1016/0006-291X(85)91946-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cherel I., Marion-Poll A., Meyer C., Rouze P. Immunological comparisons of nitrate reductase of different plant species using monoclonal antibodies. Plant Physiol. 1986 Jun;81(2):376–378. doi: 10.1104/pp.81.2.376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chérel I., Gonneau M., Meyer C., Pelsy F., Caboche M. Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia. Plant Physiol. 1990 Mar;92(3):659–665. doi: 10.1104/pp.92.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Correll C. C., Ludwig M. L., Bruns C. M., Karplus P. A. Structural prototypes for an extended family of flavoprotein reductases: comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin. Protein Sci. 1993 Dec;2(12):2112–2133. doi: 10.1002/pro.5560021212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crawford N. M., Arst H. N., Jr The molecular genetics of nitrate assimilation in fungi and plants. Annu Rev Genet. 1993;27:115–146. doi: 10.1146/annurev.ge.27.120193.000555. [DOI] [PubMed] [Google Scholar]
  9. Dailey F. A., Kuo T., Warner R. L. Pyridine nucleotide specificity of barley nitrate reductase. Plant Physiol. 1982 May;69(5):1196–1199. doi: 10.1104/pp.69.5.1196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Friemann A., Brinkmann K., Hachtel W. Sequence of a cDNA encoding the bi-specific NAD(P)H-nitrate reductase from the tree Betula pendula and identification of conserved protein regions. Mol Gen Genet. 1991 May;227(1):97–105. doi: 10.1007/BF00260713. [DOI] [PubMed] [Google Scholar]
  11. Friemann A., Lange M., Hachtel W., Brinkmann K. Induction of Nitrate Assimilatory Enzymes in the Tree Betula pendula. Plant Physiol. 1992 Jul;99(3):837–842. doi: 10.1104/pp.99.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hackett C. S., Novoa W. B., Ozols J., Strittmatter P. Identification of the essential cysteine residue of NADH-cytochrome b5 reductase. J Biol Chem. 1986 Jul 25;261(21):9854–9857. [PubMed] [Google Scholar]
  13. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  14. Hooykaas P. J., Schilperoort R. A. Agrobacterium and plant genetic engineering. Plant Mol Biol. 1992 May;19(1):15–38. doi: 10.1007/BF00015604. [DOI] [PubMed] [Google Scholar]
  15. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
  16. Karplus P. A., Daniels M. J., Herriott J. R. Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family. Science. 1991 Jan 4;251(4989):60–66. [PubMed] [Google Scholar]
  17. Lu G., Campbell W. H., Schneider G., Lindqvist Y. Crystal structure of the FAD-containing fragment of corn nitrate reductase at 2.5 A resolution: relationship to other flavoprotein reductases. Structure. 1994 Sep 15;2(9):809–821. doi: 10.1016/s0969-2126(94)00082-4. [DOI] [PubMed] [Google Scholar]
  18. McGarvey P., Kaper J. M. A simple and rapid method for screening transgenic plants using the PCR. Biotechniques. 1991 Oct;11(4):428–432. [PubMed] [Google Scholar]
  19. Meyer C., Cherel I., Moureaux T., Hoarau J., Gabard J., Rouze P. Bromphenol blue: nitrate reductase activity in Nicotiana plumbaginifolia: an immunochemical and genetic approach. Biochimie. 1987 Jun-Jul;69(6-7):735–742. doi: 10.1016/0300-9084(87)90194-5. [DOI] [PubMed] [Google Scholar]
  20. Miyazaki J., Juricek M., Angelis K., Schnorr K. M., Kleinhofs A., Warner R. L. Characterization and sequence of a novel nitrate reductase from barley. Mol Gen Genet. 1991 Sep;228(3):329–334. doi: 10.1007/BF00260624. [DOI] [PubMed] [Google Scholar]
  21. Moureaux T., Leydecker M. T., Meyer C. Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies. Eur J Biochem. 1989 Feb 15;179(3):617–620. doi: 10.1111/j.1432-1033.1989.tb14591.x. [DOI] [PubMed] [Google Scholar]
  22. Odell J. T., Nagy F., Chua N. H. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. 1985 Feb 28-Mar 6Nature. 313(6005):810–812. doi: 10.1038/313810a0. [DOI] [PubMed] [Google Scholar]
  23. Okamoto P. M., Fu Y. H., Marzluf G. A. Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover. Mol Gen Genet. 1991 Jun;227(2):213–223. doi: 10.1007/BF00259673. [DOI] [PubMed] [Google Scholar]
  24. Ostrowski J., Barber M. J., Rueger D. C., Miller B. E., Siegel L. M., Kredich N. M. Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase. J Biol Chem. 1989 Sep 25;264(27):15796–15808. [PubMed] [Google Scholar]
  25. Segal A. W., Abo A. The biochemical basis of the NADPH oxidase of phagocytes. Trends Biochem Sci. 1993 Feb;18(2):43–47. doi: 10.1016/0968-0004(93)90051-n. [DOI] [PubMed] [Google Scholar]
  26. Segal A. W., West I., Wientjes F., Nugent J. H., Chavan A. J., Haley B., Garcia R. C., Rosen H., Scrace G. Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes. Biochem J. 1992 Jun 15;284(Pt 3):781–788. doi: 10.1042/bj2840781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shirabe K., Yubisui T., Nishino T., Takeshita M. Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Cys-273 and Cys-283 are located close to the NADH-binding site but are not catalytically essential. J Biol Chem. 1991 Apr 25;266(12):7531–7536. [PubMed] [Google Scholar]
  28. Timmons T. M., Dunbar B. S. Protein blotting and immunodetection. Methods Enzymol. 1990;182:679–688. doi: 10.1016/0076-6879(90)82053-5. [DOI] [PubMed] [Google Scholar]
  29. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  30. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  31. Yubisui T., Miyata T., Iwanaga S., Tamura M., Yoshida S., Takeshita M., Nakajima H. Amino acid sequence of NADH-cytochrome b5 reductase of human erythrocytes. J Biochem. 1984 Aug;96(2):579–582. doi: 10.1093/oxfordjournals.jbchem.a134871. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES