PURIFICATION AND CHARACTERIZATION OF FUMARASE FROM THE SYNTROPHIC PROPIONATE-OXIDIZING BACTERIUM STRAIN MPOB

Fumarase from the syntrophic propionate-oxidizing bacterium strain MPO B was purified 130-fold under anoxic conditions. The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunit s of 60 kDa. The enzyme exhibited maximum activity at pH 8.5 and appro ximately 54 degrees C. The K-m 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-mercap toethanol under anoxic conditions. EPR spectroscopy of the purified en zyme revealed the presence of a [3Fe-4S] cluster. Under reducing condi tions, only a trace amount of a [4Fe-4S] cluster was detected. Additio n of fumarate resulted in a significant increase of this [4Fe-4S] sign al. The N-terminal amino acid sequence showed similarity to the sequen ces of fumarase A and B of Escherichia coli (56%) and fumarase A of Sa lmonella typhimurium (63%).