Purification and characterization of beta-methylaspartase from Fusobacterium varium.
Beta-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. Beta-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.
Bearne
S L
SL
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
White
R L
RL
MacDonnell
J E
JE
Bahrami
S
S
Grønlund
J
J
D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D000642:Ammonia-Lyases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002412:Cations / Q000494:pharmacology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003641:Deamination; D019281:Dimerization; D005673:Fusobacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D013237:Stereoisomerism
D001426:Bacterial Proteins; D002412:Cations; C018934:3-methylaspartic acid; D001224:Aspartic Acid; D000641:Ammonia; D000642:Ammonia-Lyases; C034881:methylaspartate ammonia-lyase