Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB
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© Springer-Verlag 1996
O R I G I N A L PA P E R
Bernardina L. M. Van Kuijk · Nico-Dirk Van Loo · Alexander F. Arendsen · Wilfred R. Hagen · Alfons J. M. Stams
Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB
Received: 15 September 1995 / Accepted: 13 November 1995
Abstract Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions. The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa. The enzyme exhibited maximum activity at pH 8.5 and approximately 54° C. The Km values for fumarate and L-malate were 0.25 mM and 2.38 mM, respectively. Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe2+ and β-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a [3Fe4S] cluster. Under reducing conditions, only a trace amount of a [4Fe-4S] cluster was detected. Addition of fumarate resulted in a significant increase of this [4Fe-4S] signal. The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%). Key words Fumarase · Syntrophy · Propionate oxidation · Fumarate fermentation · Anaerobic oxidation · Iron-sulfur cluster
Introduction The anaerobic oxidation of propionate to acetate, carbon dioxide, hydrogen, and/or formate is an important process in methanogenic environments (Gujer and Zehnder 1983). Under thermodynamic standard conditions, propionate oxidation is endergonic (∆G0’ = +76 kJ/mol propionate;
B. L. M. Van Kuijk · N.-D. Van Loo · A. J. M. Stams (Y) Department of Microbiology, Agricultural University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands Tel. +31-317-483101; Fax +31-317-483829 e-mail: [email protected] A. F. Arendsen · W. R. Hagen Department of Biochemistry, Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
Thauer et al. 1977). Bacteria are only able to grow by this conversion when the concentration of the products is kept low. This results in obligate syntrophic growth of propionate-oxidizing bacteria with hydrogen-, formate-, and acetate-consuming methanogens (Schink 1992; Stams 1994). Because of their syntrophic growth, little is known about the biochemistry of propionate-oxidizing bacteria. However, the propionate-oxidizing strain MPOB is able to grow in the absence of a syntrophic partner organism by the fermentation of fumarate (Plugge et al. 1993; Stams et al. 1993). Enzyme measurements and 13C-NMR experiments have indicated that the methylmalonyl-CoA pathway is involved in propionate oxidation by syntrophic cultures (Koch 1983; Houwen et al. 1987, 1990, 1991). Characteristic of this pathway is the coupling of the carboxylation of propionyl-CoA to methylmalonyl-CoA to the decarboxylation of oxaloacetate to pyruvate via a transcarboxylase. During the oxidation of propionate, reducing eq
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