Purification and characterization of a 4-hydroxybenzoate decarboxylase from an anaerobic coculture

The oxygen-sensitive 4-hydroxybenzoate decarboxylase (4OHB-DC) activity fro m 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 K-m was 5.4 mM. The activity decreased by 50% in the presence of 5 mM EDTA, and it was restored and eve n 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 chromato graphy, 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% homol ogy with the pyruvate-flavodoxin oxidoreductase (nifJ gene product) from En terobacter agglomerans and Klebsiella pneumoniae, respectively. The purifie d enzyme also slowly catalyzed the reverse reaction, that is the phenol car boxylation. These characteristics suggest that this enzyme is different fro m other known decarboxylases. This includes the 4OHB-DC from Clostridium hy droxybenzoicum, which is the only one that had been purified before.