Dihydroxybenzoate decarboxylase

A novel decarboxylase, 2,6-dihydroxybenzoate decarboxylase, was found in Agrobacterium tumefaciens 1AM 12048 at first. Thereafter, the same activity was found in Rhizobium species by two groups independently. Furthermore, Pandoraea sp. 12B-2, the most powerful producer of 2,6-dihydroxybenzoate decarboxylase, was isolated. These enzymes have been purified and characterized. 

6-Dihydroxybenzoate decarboxylase activity of these bacteria was induced specifically by 2,6-dihydroxybenzoate. The enzyme activity in a cell-free extract of A. tumefaciens 1AM 12048 was stable during storage at 4°C for 7 days in potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol. Different from 4-hydroxybenzoate decarboxylase and 3,4-dihydroxybenzoate decarboxylase, 2,6-dihydroxybenzoate decarboxylase was much less labile and barely 

When the whole-cell reaction was carried out in the presence of 0.1-3 M 1,3-dihydroxybenzene and 3M KHCO3, the molar conversion ratio of 1,3-dihydroxybenzene always reached 45-50% (Fig. 6). The high concentration of 1,3-dihydroxybenzene was not inhibitory on 2,6-dihydroxybenzoate decarboxylase. When 3M 1,3-dihydroxybenzene was incubated with the whole cells of Pandoraea sp. 12B-2 (43.9 mg as dry cell weight) in the presence of 3M KHCO3, 220mgmr of 2,6-dihydroxybenzoate (1.42 M) accumulated after 120 h incubation, with a conversion ratio of 48%. During the carboxylation of 1,3-dihydroxybenzene, no other product except for 2,6-dihydroxybenzoate was formed. 

The product was isolated and identified by H NMR and NMR analyses comparing with the authentic 2,6-dihydroxybenzoic acid as a reference. 

Carboxylation of 20-300 mM 1,2-dihydroxybenzene was carried out using 36.9 mg (as dry cell weight) of whole cells in the presence of 3M KHCO3 in 1 ml of the reaction mixture. The molar conversion yields were almost the same using 20, 100, and 200 mM 1,2-dihydroxybenzene (approximately 25%) as shown in Fig. 7. 

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Dihydroxybenzoate decarboxylase purified from Aspergillus niger has a molecular mass of 120 kDa and consists of four identical 28kDa subunits. 2,3-Dihydroxybenzoate has a value of 340 jlM and does not act on salicylate, anthranilate, 2,3-dihydroxybenzaldehyde, 2,4-dihydroxybenzoate,... 

Recently, the distribution of 2,3-dihydroxybenzoate decarboxylase has been found in a variety of fungal strains (unpubhshed data), and the carboxylation activity for catechol is confirmed by the reaction using resting cells (or cell-free extract) in the presence of 3M KHCO3. The detailed comparative studies of enzyme structures and catalytic properties between 2,3-dihydroxybenzoate decarboxylase and 3,4-dihyroxybenzoate decarboxylase might explain how the decarboxylases catalyze the regioselective carboxylation of catechol. 

Dihydroxybenzoate decarboxylase Anig 28 Unidentified hypothetical proteins... 

An enzymatic pathway for indole degradation was found in A. niger, inducible by the substrate within a 5-h period during growth. Among the enzymes found, anthranilate hydroxylase, N-formylanthranilate deformylase, 2,3-dihydroxybenzoate decarboxylase, and catechol dioxygenase were isolated, and their activities were demonstrated in a cell-free system [342],... 

Zhang et al. isolated Clostridium hydroxybenzoicum containing two inducible 4-hydroxybenzoate decarboxylase and 3,4-dihydroxybenzoate decarboxylase that form phenol and catechol (1,2-dihydroxybenzene), respectively. The organism does not further metabolize phenol and catechol produced by these reactions. The carboxylation activities of the two purified decarboxylases are not... 

A 3,4-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. 

In the aligned primary structures of class I decarboxylases, the conserved amino acid residues are scattered over their primary structures. There have been few reports to identify the amino acid residues essential for catalytic activity or substrate binding. Huang et al. reported the E-X-P motif in the alignment analysis for 4-hydroxybenzoate decarboxylase of C. hydroxybenzoicum and its homologous unidentified proteins. The E-X-P motif is also conserved in pyrrole-2-carboxylate decarboxylase and indole-3-carboxylate decarboxylase (unpublished data). However, the corresponding motif sequence is not observed in the primary structures of 3,4-dihydroxybenzoate decarboxylase of E. cloacae P241. ... 

The enzymes catalyzing the Kolbe-Schmitt carboxylation seem to occur ubiquitously. Some of them, such as 2,6-dihydroxybenzoate decarboxylase and pyrrole-2-carboxylate decarboxylase, catalyze efficiently the reverse carboxylation reaction and accumulate high concentration of 2,6-dihydroxybenzoate from 1,3-dihydroxybenzene and pyrrole-2-carboxylate from pyrrole, respectively, in the... 

He, Z. and J. Wiegel. 1996. Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxyben-zoicum.. Bacteriol. 178 3539-3543. 

Various enzymes are known to catalyze arene carboxylation or decarboxylation in nature [41]. The first report [42] of a preparatively useful biocatalytic arene carboxylation was the para-carboxylation of phenol using the enzyme phenyl phosphate carboxylase. As the name imphes, this requires the phenol to be phosphorylated prior to reaction. Subsequently, phenol para-carboxylation without prior phosphorylation was demonstrated using enzymes such as 4-hydroxybenzoate decarboxylase [43-45] and 3,4-dihydroxybenzoate decarboxylase [46, 47] (Scheme 32.10). Although the natural function of these latter enzymes is to catalyze catalyze reactions in either direction, dependent on the reaction conditions. 

The reversible decarboxylation of 4-hydroxybenzoate and 3,4-dihydroxybenzoate has been described in Sedimentibacter (Clostridium) hydroxybenzoicum (He and Wiegel 1996), and the oxygen-sensitive enzyme has been purified. The decarboxylase from Pantoae... 

Goto M, Hayashi H, Miyahara I, Hirotsu K, Yoshida M, Oikawa T (2006) Crystal structmes of nonoxidative Zn-dependent 2,6-dihydroxybenzoate (y-resorcylate) decarboxylase from Rhizobium sp. strain Mtp-10005. J Biol Chem 281 34365-34373... 

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