Dihydroxybenzoate decarboxylase EC

4-dihydroxybenzoate decarboxylase (EC 4.1.1.63) was purified from C. hydroxybenzoicum and characterized for the first time. The estimated molecular mass of the enzyme is 270 kDa. The subunit molecular mass is 57kDa, suggesting that the enzyme consists of five identical subunits. The temperature and pH optima are 50°C and pH 7.0, respectively. The Arrhenius energy for decarboxylation of 3,4-dihydroxybenzoate was 32.5 kJ mol for the temperature range from 22 to 50°C. The and for 3,4-dihydroxybenzoate were 0.6 mM and 5.4 X 10 min respectively, at pH 7.0 and 25°C. The enzyme catalyzes the reverse reaction, that is, the carboxylation of catechol to 3,4-dihydroxybenzoate, at pH 7.0. The enzyme does not decarboxylate 4-hydroxybenzoate. Although the equilibrium of the reaction is on the side of catechol, it is postulated that C. hydroxybenzoicum uses the enzyme to convert catechol to 3,4-dihydroxybenzoate.  

The occurrence of 3,4-dihydroxybenzoate decarboxylase was also found widely in facultative anaerobes. Among them, Enterobacter cloacae P241 showed the highest activity of 3,4-hydroxybenzoate decarboxylase, and the activity of the cell-free extract of E. cloacae P241 was determined to be 0.629 p.mol min (mg protein) at 30°C, which was more than that of C. hydroxybenzoicum, 0.11 (xmol min mg protein) at 25°C. The E. cloacae P241 enzyme has a molecular mass of 334 kDa and consists of six identical 50 kDa subunits. The value for 3,4-dihydroxybenzoate was 177 p.M. The enzyme is also characteristic of its narrow substrate specificity and does not act on 4-hydroxybenzoate and other benzoate derivatives. The properties of E. cloacae P241 3,4-hydroxybenzoate decarboxylase were similar to those of C. hydroxybenzoicum in optimum temperature and pH, oxygen sensitivity, and substrate specificity. 

The reaction product of the reserve carboxylation reaction was isolated and identified to be 3,4-dihydroxybenzoic acid by NMR and NMR with the authentic 3,4-dihydroxybenzoic acid as a reference. The carboxylation reaction of catechol to 3,4-dihydroxybenzoate was affected by the concentration of KHCO3. The carboxylation activity of E. cloacae P241 3,4-dihydroxybenzoate decarboxylase in the presence of 0.1 M KHCO3 was only 15% of that in the presence of 3 M KHC03. In the case of C. hydroxybenzoicum 3,4-dihydroxybenzote decarboxylase, only 0.01 mM 3,4-dihydroxybenzoate was formed from 6mM catechol in the presence of 50 mM NaHC03 by 40 min incubation. The difference in molar conversion ratios might be caused by the concentration of bicarbonate added to the reaction mixture. 

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