Purification and characterization of beta-methylaspartase from Fusobacterium varium

beta -Methylaspartase (EC 4.3.1.2) was purified 20-fold in 35% yield from F usobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAE-Sepharose. The enz yme is dimeric, consisting of two identical 46 kDa subunits, and requires M g2+ (K-m = 0.27 +/- 0.01 mM) and K+ (K-m = 3.3 +/- 0.8 mM) for maximum acti vity. beta -Methylaspartase-catalyzed addition of ammonia to mesaconate yie lded two diastereomeric amino acids, identified by HPLC as (2S,3S)-3-methyl aspartate (major product) and (2S,3R)-3-methylaspartate (minor product). Op timal activity for the deamination of (2S,3S)-3-methylaspartate (K-m = 0.51 +/- 0.04 mM) was observed at pH 9.7. The N-terminal protein sequence (30 r esidues) of the F. varium enzyme is 83% identical to the corresponding sequ ence of the clostridial enzyme.