FUNCTIONAL-ANALYSIS OF THE CYSTEINE MOTIFS IN THE FERREDOXIN-LIKE PROTEIN FDXN OF RHIZOBIUM-MELILOTI INVOLVED IN SYMBIOTIC NITROGEN-FIXATION

MASEPOHL B, KUTSCHE M, RIEDEL KU, SCHMEHL M, KLIPP W, Pühler A (1992)
MOLECULAR & GENERAL GENETICS 233(1-2): 33-41.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Abstract / Bemerkung
The Rhizobium meliloti fdxN gene, which is part of the nifA-nifB-fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix- phenotype of an R. meliloti fdxN::[Tc] mutant could be rescued by the R. leguminosarum fdxN gene, whereas no complementation was observed with nif-associated genes encoding ferredoxins from Bradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum and Rhodobacter capsulatus. In addition to these heterologous genes, several R. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located downstream of this motif (position 61), was found to be essential for the activity of the R. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X2-C-X2-C-X3-C, nif-associated ferredoxins, including R. meliloti FdxN, are characterized by two different cysteine motifs. Six "additional" amino acids separate the second (Cys42) and the third cysteine (Cys51) in the C-terminal motif (C-X2-C-X8-C-X3-C). By molecular modelling, it was predicted that these amino acid residues form a loop, which does not alter the relative positions of the neighbouring cysteines. Deletion of this loop resulted in an R. meliloti FdxN mutant protein, which exhibited almost 70% wild-type activity, indicating that the predicted loop does not affect Fe-S cluster binding and plays no crucial role in activity of the FdxN protein.
Stichworte
HETEROLOGOUS COMPLEMENTATION; MOLECULAR; MODELING; SITE-DIRECTED MUTAGENESIS
Erscheinungsjahr
1992
Zeitschriftentitel
MOLECULAR & GENERAL GENETICS
Band
233
Ausgabe
1-2
Seite(n)
33-41
ISSN
0026-8925
eISSN
1617-4623
Page URI
https://pub.uni-bielefeld.de/record/1648216

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MASEPOHL B, KUTSCHE M, RIEDEL KU, SCHMEHL M, KLIPP W, Pühler A. FUNCTIONAL-ANALYSIS OF THE CYSTEINE MOTIFS IN THE FERREDOXIN-LIKE PROTEIN FDXN OF RHIZOBIUM-MELILOTI INVOLVED IN SYMBIOTIC NITROGEN-FIXATION. MOLECULAR & GENERAL GENETICS. 1992;233(1-2):33-41.
MASEPOHL, B., KUTSCHE, M., RIEDEL, K. U., SCHMEHL, M., KLIPP, W., & Pühler, A. (1992). FUNCTIONAL-ANALYSIS OF THE CYSTEINE MOTIFS IN THE FERREDOXIN-LIKE PROTEIN FDXN OF RHIZOBIUM-MELILOTI INVOLVED IN SYMBIOTIC NITROGEN-FIXATION. MOLECULAR & GENERAL GENETICS, 233(1-2), 33-41. doi:10.1007/BF00587558
MASEPOHL, B., KUTSCHE, M., RIEDEL, K. U., SCHMEHL, M., KLIPP, W., and Pühler, A. (1992). FUNCTIONAL-ANALYSIS OF THE CYSTEINE MOTIFS IN THE FERREDOXIN-LIKE PROTEIN FDXN OF RHIZOBIUM-MELILOTI INVOLVED IN SYMBIOTIC NITROGEN-FIXATION. MOLECULAR & GENERAL GENETICS 233, 33-41.
MASEPOHL, B., et al., 1992. FUNCTIONAL-ANALYSIS OF THE CYSTEINE MOTIFS IN THE FERREDOXIN-LIKE PROTEIN FDXN OF RHIZOBIUM-MELILOTI INVOLVED IN SYMBIOTIC NITROGEN-FIXATION. MOLECULAR & GENERAL GENETICS, 233(1-2), p 33-41.
B. MASEPOHL, et al., “FUNCTIONAL-ANALYSIS OF THE CYSTEINE MOTIFS IN THE FERREDOXIN-LIKE PROTEIN FDXN OF RHIZOBIUM-MELILOTI INVOLVED IN SYMBIOTIC NITROGEN-FIXATION”, MOLECULAR & GENERAL GENETICS, vol. 233, 1992, pp. 33-41.
MASEPOHL, B., KUTSCHE, M., RIEDEL, K.U., SCHMEHL, M., KLIPP, W., Pühler, A.: FUNCTIONAL-ANALYSIS OF THE CYSTEINE MOTIFS IN THE FERREDOXIN-LIKE PROTEIN FDXN OF RHIZOBIUM-MELILOTI INVOLVED IN SYMBIOTIC NITROGEN-FIXATION. MOLECULAR & GENERAL GENETICS. 233, 33-41 (1992).
MASEPOHL, B, KUTSCHE, M, RIEDEL, KU, SCHMEHL, M, KLIPP, W, and Pühler, Alfred. “FUNCTIONAL-ANALYSIS OF THE CYSTEINE MOTIFS IN THE FERREDOXIN-LIKE PROTEIN FDXN OF RHIZOBIUM-MELILOTI INVOLVED IN SYMBIOTIC NITROGEN-FIXATION”. MOLECULAR & GENERAL GENETICS 233.1-2 (1992): 33-41.

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