Skip to main content
SpringerLink
Log in

Purification and properties of the formate dehydrogenase and characterization of the fdhA gene of Sulfurospirillum multivorans

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene (fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe4/S4 cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

Fdh :

Formate dehydrogenase

PCE :

Tetrachloroethene

References

  • Almendra MJ, Brondino CD, Gavel O, Pereira AS, Tavares P, Bursakov S, Duarte R, Caldeira J, Moura JJG, Moura I (1999) Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas. Biochemistry 38:16366–16372

    Article  CAS  PubMed  Google Scholar 

  • Altschul SF, Madden TL, Schaeffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402.

    PubMed  Google Scholar 

  • Bokranz M, Gutmann, M, Koertner C, Kojro E., Fahrenholz F, Lauterbach F, Kröger A (1991) Cloning and nucleotide sequence of the structural genes encoding the formate dehydrogenase of Wolinella succinogenes. Arch Microbiol 156:119–128

    CAS  PubMed  Google Scholar 

  • Boyington JC, Gladyshev VN, Khangulov SV, Stadtman TC, Sun PD (1997) Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4/S4 cluster. Science 275:1305–1308

    CAS  PubMed  Google Scholar 

  • de Bok FAM, Hagedoorn P-L, Silva PJ, Hagen WR, Schiltz E, Fritsche K, Stams AJM (2003) Two W-containing formate dehydrogenases (CO2-reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidans. Eur J Biochem 270:2476–2485

    Article  PubMed  Google Scholar 

  • Graentzdoerffer A, Rauh D, Pich A, Andreesen JR (2003) Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase. Arch Microbiol 179:116–130

    CAS  PubMed  Google Scholar 

  • Hille R, Rétey J, Bartlewski-Hof U, Reichenbecher W, Schink B (1999) Mechanistic aspects of molybdenum-containing enzymes. FEMS Microbiol Rev 22:489–501

    Article  Google Scholar 

  • Holliger C, Wohlfahrt G, Diekert G (1999) Reductive dechlorination in the energy metabolism of anaerobic bacteria. FEMS Microbiol Rev 22:383–398

    Article  Google Scholar 

  • Johnson JL, Bastian NR, Rajagopalan, KV (1990) Molybdopterin guanine dinucleotide: a modified form of molybdopterin identified in the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides forma specialis denitrificans. Proc Natl Acad Sci USA 77:5172–5176

    Google Scholar 

  • Jormakka M, Tornroth S, Byrne B, Iwata S (2002) Molecular basis of proton motive force generation: structure of formate dehydrogenase-N. Science 295:1863–1868

    PubMed  Google Scholar 

  • Khangulov SV, Gladyshev VN, Dismukes GC, Stadtman TC (1998) Selenium-containing formate dehydrogenase H from Escherichia coli: a molybdopterin enzyme that catalyzes formate oxidation without oxygen transfer. Biochemistry 37:3518–3528

    Article  CAS  PubMed  Google Scholar 

  • Kröger A, Winkler E, Innerhofer A, Hackenberg H, Schägger H (1979) The formate dehydrogenase involved in electron transport from formate to fumarate in Vibrio succinogenes. Eur J Biochem 94:465–475

    PubMed  Google Scholar 

  • Kyte J, Doolittle RF (1982) A simple method for displaying the hydrophatic character of a protein. J Mol Biol 157:105–132

    CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Laukel M, Chistoserdova L, Lidstrom ME, Vorholt JA (2003) The tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1: Purification and properties. Eur J Biochem 270:325–333

    Article  CAS  PubMed  Google Scholar 

  • Lenger R, Herrmann U, Gross R, Simon J, Kröger A (1997) Structure and function of a second gene cluster encoding the formate dehydrogenase of Wolinella succinogenes. Eur J Biochem 246:646–651

    CAS  PubMed  Google Scholar 

  • Liu C-L, Mortenson LE (1984) Formate dehydrogenase of Clostridium pasteurianum. J Bacteriol 159:375–380

    CAS  PubMed  Google Scholar 

  • Liu Z, Reches M, Groisman, I, Engelberg-Kulka H (1998) The nature of the minimal ‘selenocysteine insertion sequence’ (SECIS) in Escherichia coli. Nucleic Acids Res 26:896–902

    Article  CAS  PubMed  Google Scholar 

  • Luijten MLGC, de Weert J, Smidt H, Boschker HTS, de Vos WM, Schraa G, Stams AJM (2003) Description of Sulfurospirillum halorespirans sp. nov., an anaerobic, tetrachloroethene-respiring bacterium, and transfer of Dehalospirillum multivorans to the genus Sulfurospirillum as Sulfurospirillum multivorans comb. nov. Int J Syst Bacteriol 53: 787–793

    Article  CAS  Google Scholar 

  • Magalon A, Asso M, Guigliarelli B, Rothery RA, Bertrand P, Giordano G, Blasco F (1998) Molybdenum cofactor properties and [FeS] cluster coordination in Escherichia coli nitrate reductase A: Investigation by site-directed mutagenesis of the conserved His-50 residue in the NarG subunit. Biochemistry 37:7363–7370

    CAS  PubMed  Google Scholar 

  • Miller E, Wohlfarth G, Diekert G (1997) Studies on tetrachloroethene respiration in Dehalospirillum multivorans. Arch Microbiol 166:379–387

    Article  Google Scholar 

  • Neumann A, Scholz-Muramatsu H, Diekert G (1994) Tetrachloroethene metabolism of Dehalospirillum multivorans. Arch Microbiol 162:295–301

    CAS  PubMed  Google Scholar 

  • Neumann A, Wohlfarth G, Diekert G (1996) Purification and characterization of tetrachloroethene reductive dehalogenase from Dehalospirillum multivorans. J Biol Chem 271:16515–16519

    CAS  PubMed  Google Scholar 

  • Neumann A, Wohlfarth G, Diekert G (1998) Tetrachloroethene dehalogenase from Dehalospirillum multivorans: Cloning, sequencing of the encoding genes, and expression of the pceA gene in Escherichia coli. J Bacteriol 180:4140–4145

    CAS  PubMed  Google Scholar 

  • Oh JI, Bowien B (1998) Structural analysis of the fds operon encoding the NAD+-linked formate dehydrogenase of Ralstonia eutropha. J Biol Chem 273:26349–60

    Article  CAS  PubMed  Google Scholar 

  • Parkhill J, Wren BW, Mungall K, Ketley JM, Churcher C, Basham D, Chillingworth T, Davies RM, Feltwell T, Holroyd S, Jagels K, Karlyshev AV, Moule S, Pallen MJ, Penn CW, Quail MA, Rajandream MA, Rutherford KM, van Vliet AH, Whitehead S, Barrell BG (2000) The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665–668

    Article  CAS  PubMed  Google Scholar 

  • Raaijmakers H, Macieira S, Dias JM, Teixeira S, Bursakov S, Huber R, Moura JJ, Moura I, Romao MJ (2002) Gene sequence and the 1.8 A crystal structure of the tungsten-containing formate dehydrogenase from Desulfovibrio gigas. Structure 10:1261–72

    Article  CAS  PubMed  Google Scholar 

  • Rose TM, Schultz ER, Henikoff JG, Pietrokovski S, McCallum CM, Henikoff S (1998) Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly-related sequences. Nucleic Acids Res 26:1628–1635

    Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual, 3. edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

  • Sargent F, Berks BC, Palmer T (2002) Assembly of membrane-bound respiratory complexes by the Tat protein-transport system. Arch Microbiol 178:77–84

    Article  CAS  PubMed  Google Scholar 

  • Schauer NL, Ferry JG (1982) Properties of a formate dehydrogenase in Methanobacterium formicicum. J Bacteriol 50:1–7

    Google Scholar 

  • Scholz-Muramatsu H, Neumann A, Meßmer M, Moore E, Diekert G (1995) Isolation and characterization of Dehalospirillum multivorans gen. nov. sp. nov., a tetrachloroethene-utilizing, strictly anaerobic bacterium. Arch Microbiol 163:48–56

    Article  CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    PubMed  Google Scholar 

  • Voordouw G (2000) A universal system for the transport of redox proteins: early roots and latest developments. Biophys Chem 86:131–140

    CAS  PubMed  Google Scholar 

  • Wohlfarth G, Diekert G (1999) Anaerobic dehalogenases. Curr Opin Biotechnol 8:290–295

    Article  Google Scholar 

  • Zehnder AJB, Wuhrmann K (1976) Titanium (III) citrate as a non-toxic, oxidation-reduction buffering system for the culture of obligate anaerobes. Science 194:1165–1166

    CAS  PubMed  Google Scholar 

  • Zinoni F, Birkmann A, Leinfelder W, Böck A (1987) Cotranslational insertion of selenocysteine into formate dehydrogenase from E. coli directed by a UGA codon. Proc Natl Acad Sci USA 84:3156–3160

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Deutsche Forschungsgemeinschaft, the EC, and the Fonds der Chemischen Industrie. The authors would like to thank P. Brand for skillful technical assistance, and Dr. K. Voigt (Dept. of General Microbiology and Microbe Genetics, FSU Jena, Germany) for gene sequencing and helpful discussions.

Author information

Authors and Affiliations

  1. Institut für Mikrobiologie, FSU Jena, Philosophenweg 12, Lehrstuhl für Angewandte und Ökologische Mikrobiologie, 07743 , Jena, Germany

    Roland P. H. Schmitz & Gabriele Diekert

Authors
  1. Roland P. H. Schmitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gabriele Diekert
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Roland P. H. Schmitz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schmitz, R.P.H., Diekert, G. Purification and properties of the formate dehydrogenase and characterization of the fdhA gene of Sulfurospirillum multivorans . Arch Microbiol 180, 394–401 (2003). https://doi.org/10.1007/s00203-003-0604-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-003-0604-x

Keywords

Search

Navigation