, Volume 221, Issue 1-2, pp 117-126

Purification and characterization of β‐methylaspartase from Fusobacterium varium

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Abstract

β‐Methylaspartase (EC 4.3.1.2) was purified 20‐fold in 35% yield from Fusobacterium 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 enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg2+ (Km = 0.27 ± 0.01 mM) and K+ (Km = 3.3 ± 0.8 mM) for maximum activity. β‐Methylaspartase‐catalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)‐3‐methylaspartate (major product) and (2S,3R)‐3‐methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)‐3‐methylaspartate (Km = 0.51 ± 0.04 mM) was observed at pH 9.7. The N‐terminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.