Preferences help
enabled [disable] Abstract
Number of results
2008 | 55 | 4 | 753-760
Article title

Mass spectrometry identification of membrane-bound respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus)

Title variants
Languages of publication
Respiratory nitrate reductase (NR) from Bradyrhizobium sp. (Lupinus) USDA 3045 has biochemical properties of the membrane-bound NR type. However, in the completely sequenced rhizobium genomes only genes for the periplasmic type of dissimilatory NR were found. Therefore purification and identification of the enzyme by tandem mass spectrometry (MS/MS) was undertaken. MS/MS spectra representing 149 unique tryptic peptides derived from purified 137-kDa subunit matched the NCBInr-deposited NarG sequences. MS/MS sequencing of two other SDS/PAGE bands (65- and 59-kDa) identified them as derivatives of the NarH-type protein. Applying additional validation criteria, 73% of the sequence of the NarG subunit (902 aa) and 52% of NarH sequence (266 aa) was assembled (UniProt KB acc. no. P85097 and P85098). This is the first unambiguous identification of an active NarGH-like NR in rhizobia. Moreover, arguments are provided here for the existence of a functional enzyme of this type also among other rhizobial species, basing on immunoblot screening and the presence of membrane-associated NR-active electrophoretic forms.

Physical description
  • Institute of Experimental Biology, Adam Mickiewicz University, Poznań, Poland
  • Bell LC, Richardson DJ, Ferguson SJ (1990) Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha. FEBS Lett 265: 85-87.
  • Berks BC, Ferguson SJ, Moir JW, Richardson DJ (1995) Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim Biophys Acta 1232: 97-173.
  • Bertero MG, Rothery RA, Palak M, Hou C, Lim D, Blasco F, Weiner JH, Strynadka NCJ (2003) Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A. Nature Struct Biol 10: 681-687.
  • Blasco F, Guigliarelli B, Magalon A, Asso M, Giordano G, Rothery RA (2001) The coordination and function of the redox centres of the membrane-bound nitrate reductases. Cell Mol Life Sci 58: 179-193.
  • Blümle S, Zumft WG (1991) Respiratory nitrate reductase from denitrifying Pseudomonas stutzeri, purification, properties and target of proteolysis. Biochim Biophys Acta 1057: 102-108.
  • Clamp M, Cuff J, Searle SM, Barton GJ (2004) The Jalview Java alignment editor. Bioinformatics 20: 426-427.
  • Delgado MJ, Bonnard N, Tresierra-Ayala A, Bedmar EJ, Muller P (2003) The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration. Microbiology 149: 3395-3403.
  • Galibert F, Finan TM, Long SR et al. (2001) The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293: 668-672.
  • González PJ, Correia C, Moura I, Brondino CD, Moura JJG (2006a) Bacterial nitrate reductases. Molecular and biological aspects of nitrate reduction. J Inorg Biochem 100: 1015-1023.
  • González V, Santamaria RI, Bustos P et al. (2006b) The partitioned Rhizobium etli genome: Genetic and metabolic redundancy in seven interacting replicons. Proc Natl Acad Sci USA 103: 3834-3839.
  • Hettmann T, Anemuller S, Borcherding H, Mathe L, Steinrucke P, Diekmann S (2003) Pseudomonas stutzeri soluble nitrate reductase αβ-subunit is a soluble enzyme with a similar electronic structure at the active site as the inner membrane-bound αβγ holoenzyme. FEBS Lett 534: 143-150.
  • Ishihama Y (2005) Proteomic LC-MS systems using nanoscale liquid chromatography with tandem mass spectrometry. J Chromatogr A 1067: 73-83.
  • Jensen ON, Larsen MR, Roepstorff P (1998) Mass spectrometric identification and microcharacterization of proteins from electrophoretic gels: strategies and applications Proteins (Suppl) 2: 74-89.
  • Jormakka M, Richardson D, Byrne B, Iwata S (2004) Architecture of NarGH reveals a structural classification of Mo-bisMGD enzymes. Structure 12: 95-104.
  • Kaneko T, Nakamura Y, Sato S et al. (2000) Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res 7: 331-338.
  • Kaneko T, Nakamura Y, Sato S et al. (2002) Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Res 9: 189-197.
  • Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680-685.
  • Lopez R, Silventoinen V, Robinson S, Kibria A, Gish W (2003) WU-Blast2 server at the European Bioinformatics Institute. Nucleic Acids Res 31: 3795-3798.
  • Medzihradszky KF (2005) Peptide sequence analysis. Methods Enzymol 402: 209-244.
  • Millipore (2000) Rapid immunodetection of blotted proteins without blocking -Immobilon-P transfer membrane. Application Note RP562.
  • Moreno-Vivián C, Ferguson SJ (1998) Definition and distinction between assimilatory dissimilatory and respiratory pathways. Mol Microbiol 29: 664-666.
  • Nesvizhskii AJ, Aebersold A (2004) Analysis statistical validation and dissemination of large-scale proteomics datasets generated by tandem MS. Drug Discov Today 9: 173-181.
  • Perkins DN, Pappin DJC, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551-3567.
  • Philippot L, Højberg O (1999) Dissimilatory nitrate reductases in bacteria. Biochim Biophys Acta 1446: 1-23.
  • Polcyn W (2008) Nitrate-related down-regulation of respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus). Acta Biochim Polon 55: 761-766.
  • Polcyn W, Luciński R (2003) Aerobic and anaerobic nitrate and nitrite reduction in free-living cells of Bradyrhizobium sp (Lupinus). FEMS Microbiol Lett 226: 331-337.
  • Polcyn W, Luciński R (2006) Dissimilatory nitrate reductase from Bradyrhizobium sp (Lupinus): subcellular location, catalytic properties and characterization of the active enzyme forms. Curr Microbiol 52: 231-237.
  • Richardson DJ, Berks BC, Russell DA, Spiro S, Taylor CJ (2001) Functional, biochemical and genetic diversity of prokaryotic nitrate reductases. Cell Mol Life Sci 58: 165-178.
  • Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68: 850-858.
  • Somasegaran P, Hoben HJ (1994) Handbook for Rhizobia: Methods in Legume-Rhizobium Technology. Springer, New York, pp. 366-369.
  • Willems A (2006) The taxonomy of rhizobia: an overview. Plant Soil 287: 3-14.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.