Purification and characterization of a 4-hydroxybenzoate decarboxylase from an anaerobic coculture.
The oxygen-sensitive 4-hydroxybenzoate decarboxylase (4OHB-DC) activity from a phenol-carboxylating coculture, consisting of Clostridium-like strain 6 and an unidentified strain 7, was studied. Assays done with cell extracts showed that the optimal pH was 5.0-6.5 and the Km was 5.4 mM. The activity decreased by 50% in the presence of 5 mM EDTA, and it was restored and even enhanced by the addition of Mg++, Mn++, Zn++, or Ca++. After purification, the molecular mass of the enzyme was estimated as 420 kDa by gel chromatography, and as 119 kDa by SDS-PAGE, suggesting a homotetrameric structure. Its pI was 5.6. The N-terminal amino acid sequence showed 95% and 76% homology with the pyruvate-flavodoxin oxidoreductase (nifJ gene product) from Enterobacter agglomerans and Klebsiella pneumoniae, respectively. The purified enzyme also slowly catalyzed the reverse reaction, that is the phenol carboxylation. These characteristics suggest that this enzyme is different from other known decarboxylases. This includes the 4OHB-DC from Clostridium hydroxybenzoicum, which is the only one that had been purified before.
Li
T
T
INRS-Institut Armand-Frappier-Microbiologie et Biotechnologie, Université du Québec, Laval, Canada.
Juteau
P
P
Beaudet
R
R
Lépine
F
F
Villemur
R
R
Bisaillon
J G
JG
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000254:growth & development*; D002262:Carboxy-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology* / Q000254:growth & development*; D003470:Culture Media; D008969:Molecular Sequence Data; D010636:Phenols / Q000378:metabolism
D003470:Culture Media; D010636:Phenols; D002262:Carboxy-Lyases; C065828:4-hydroxybenzoate decarboxylase