Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB.
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 degrees 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 &beta;-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a [3Fe-4S] 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%).
Van Kuijk
B L
BL
Department of Microbiology, Agricultural University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands.
Van Loo
N D
ND
Arendsen
A F
AF
Hagen
W R
WR
Stams
A J
AJ
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D005649:Fumarate Hydratase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005650:Fumarates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology; D011422:Propionates / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D013696:Temperature
D005650:Fumarates; D008293:Malates; D011422:Propionates; C030298:malic acid; C032005:fumaric acid; D005649:Fumarate Hydratase; D010100:Oxygen