Hydroxybenzoate decarboxylase EC 41161

4-Hydroxybenzoate decarboxylase (EC 4.1.1.61) of anaerobe C. hydroxyben-zoicum was purified and characterized for the first time.20 It has an apparent molecular mass of 350 kDa and consists of six identical subunits of 57 kDa. The temperature optimum for the decarboxylation is approximately 50°C, the optimum pH being 5.6-6.2. The activation energy for decarboxylation of 4-hydroxybenzoate is 65kJmol-1 (20-37°C). The enzyme also catalyzes the decarboxylation of 3,4-dihydroxybenzoate. The apparent Km and kcat values for 4-hydroxybenzoate are 0.40 mM and 3.3 x 103 min-1, and for 3,4-dihydroxybenzoate 1.2 mM and 1.1 x 103 min-1, respectively, at pH 6.0 and 25°C. The enzyme activity was not influenced by the addition of biotin or avidin. Secondly, 4-hydroxybenzoate decar-boxylase from an anaerobic co-culture, consisting of Clostridium-like strain and an unidentified strain, was purified and characterized.27

The occurrence of 4-hydroxybenzoate decarboxylase was also found in facultative anaerobic bacteria, E. cloacae P240, and the enzyme was purified and characterized.25 The activity of the cell-free extract of E. cloacae P240 was determined to be 13.7 (xmol min-1(mg protein)-1 at 30°C, which was much higher than

COOH

COOH

Scheme 1

that of C. hydroxybenzoicum, 0.33 (xmol min-1(mg protein)-1 at 25°C.20 The purified enzyme of E. cloacae P240 is a homohexamer of identical 60 kDa subunits, which is in good agreement with that of C. hydroxybenzoicum. The properties of 4-hydroxybenzoate decarboxylase from E. cloacae P240 are similar to those of Clostridium strains in optimum temperature and pH, oxygen sensitivity, and substrate specificity.

4-Hydroxybenzoate decarboxylase catalyzes the reverse reactions, that is, the carboxylation of phenol to 4-hydroxybenzoate and of catechol to 3,4-dihydroxybenzoate.2025 Using the E. cloacae P240 purified enzyme, the carboxylation reaction of phenol was investigated in a tightly sealed reaction vessel to avoid the leakage of CO2 gas. The reaction mixture contained 20 mM phenol, 3 M KHCO3, 10 mM dithiothreitol, 100 mM potassium phosphate buffer (pH 7.0), and enzyme solution (or whole cells). The reaction was started by the addition of KHCO3 and incubated at 20°C. The decarboxylase catalyzed the reverse carboxylation reaction of 20 mM phenol to form 3.8 mM 4-hydroxybenzoate with a molar conversion yield of 19% (Fig. 1). The Km value for phenol was calculated to be 14.8 mM. The conversion ratio of phenol to 4-hydroxybezoate by C. hydroxybenzoicum decarboxylase was 0.5% in the presence of 3.3 mM phenol and 100 mM NaHCO3 by 2 h incubation; 17 jxM 4-hydroxybenzoate was formed.20 The difference in molar conversion ratios might be caused by the concentration of bicarbonate to the reaction mixture. The reversible conversion of 4-hydroxybenzoate and phenol was also observed by whole-cell suspensions. Assuming that HCO3 -is the co-substrate, the equilibrium constant calculated for the reaction 4-hydroxybenzoate-+ H2O ^ phenol + HCO- was 11.4 (± 0.5). The equilibrium constants explain the low conversion of phenol into 4-hydroxybenzoate.28

Time (min)

Figure 1: Time course of carboxylation reaction of phenol. Closed circles, 4-hydroxybenzoate; open squares, phenol.

Time (min)

Figure 1: Time course of carboxylation reaction of phenol. Closed circles, 4-hydroxybenzoate; open squares, phenol.

2.2. 3,4-Dihydroxybenzoate decarboxylase (EC 4.1.1.63)

Scheme 2

Scheme 2

A 3,4-dihydroxybenzoate decarboxylase (EC 4.1.1.63) was purified from C. hydroxybenzoicum and characterized for the first time.21 The estimated molecular mass of the enzyme is 270 kDa. The subunit molecular mass is 57 kDa, 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-1 for the temperature range from 22 to 50°C. The Km and kcat for 3,4-dihydroxybenzoate were 0.6 mM and 5.4 x 103 min-1, 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. hydrox-ybenzoicum uses the enzyme to convert catechol to 3,4-dihydroxybenzoate.21

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,26 and the activity of the cell-free extract of E. cloacae P241 was determined to be 0.629 jxmol min-1(mg protein)-1 at 30°C, which was more than that of C. hydroxybenzoicum, 0.11 (xmol min-1(mg protein)-1 at 25°C.21 The E. cloacae P241 enzyme has a molecular mass of 334 kDa and consists of six identical 50 kDa subunits. The Km value for 3,4-dihydroxybenzoate was 177 jxM. 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 carboxylation reaction of catechol by E. cloacae P241 was investigated in a tightly sealed vessel to avoid the leakage of CO2 gas.26 The reaction mixture contained 25 mM catechol, 3 M KHCO3, 10 mM dithiothreitol, 100 mM potassium phosphate buffer (pH 7.0), and enzyme solution. The reaction was started by the addition of KHCO3, incubated at 20°C. The carboxylation for catechol was also observed using the purified enzyme and the whole-cell suspension of E. cloacae P241 in the presence of 25 mM catechol and 3 M KHCO3, resulting in the formation of 5.49 mM 3,4-dihydroxybenzoate with a molar conversion ratio of 22% (Fig. 2).

Time (min)

Figure 2: Time course of carboxylation reaction of catechol. Closed circles, 3,4-dihydroxybenzoate; open squares, catechol.

Time (min)

Figure 2: Time course of carboxylation reaction of catechol. Closed circles, 3,4-dihydroxybenzoate; open squares, catechol.

The reaction product of the reserve carboxylation reaction was isolated and identified to be 3,4-dihydroxybenzoic acid by NMR and 13C 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 KHCO3.26 In the case of C. hydroxybenzoicum 3,4-dihydroxybenzote decarboxylase, only 0.01 mM 3,4-dihydroxybenzoate was formed from 6 mM catechol in the presence of 50 mM NaHCO3 by 40 min incubation.21 The difference in molar conversion ratios might be caused by the concentration of bicarbonate added to the reaction mixture.

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