Pyrrole-2-carboxylate decarboxylase

The enzyme was purified from B. megaterium PYR2910. The enzyme has a molecular mass of approximately 98 kDa and consists of two identical subunits. The values for decarboxylation were determined to be 989 units mg  

Pyrrole-2-carboxylate decarboxylase attains equilibrium in the course of either decarboxylation or carboxylation (Fig. 8). The decarboxylation of 100 mM pyrrole-2-carboxylate was in equilibrium after Ih, resulting in an equilibrium constant of 0.3 M. Due to this balanced equilibrium, the enzyme also catalyzed the reverse carboxylation of pyrrole after the addition of HCO3, leading to a similar equilibrium constant of 0.4 M and a shift of the [pyrrole]/[pyrrole-2-carboxylate] ratio toward the acid. 

The development of CO2 fixation reactions in supercritical CO2 attracts increasing attention due to its gas-like low viscosities and high diffusivities and its liquid-like solubilizing power. Matsuda et al. attempted to carry out the con- 

Pyrrole-2-carboxylate is employed in the synthesis of various pharmaceuticals and a potential herbicide. A number of organic syntheses have been described However, they require multiple steps and result in low yields. Furthermore, the chemical carbonation of pyrrole with K2CO3 depends on high pressure and temperature. The one-step enzymatic conversion has advantages with regard to regiospecificity, yield, and mild reaction conditions. 

When a cell extract prepared from A. nicotianae FI1612 cells was stored without the addition of sulfhydryl-protecting reagents, 80% of the initial activity was lost after storage at 4°C for 4 days. The enzyme activity was stabilized 

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Non-oxidafive novel aromatic acid decarboxylases, pyrrole-2-carboxylate decarboxylase and indole-3-carboxylate decarboxylase, were found in Bacillus... 

Figure 8 Decarboxylation of pyrrole-2-carboxylate (a) and carboxylation of pyrrole (b) by pyrrole-2-carboxylate decarboxylase.

The carboxylafion of indole into indole-3-carboxylate was observed by the purified indole-3-carboxylate decarboxylase as well as by the whole cells. For the carboxylafion reaction, temperatures over 30°C were not appropriate. The activities at 10, 20, and 30°C were about the same. The activity was maximal at pH 8.0 (Tris-HCl buffer, 100 mM). As shown in Fig. 10, the resting cells of A. nicotianae F11612 also catalyzed the carboxylafion of indole efficiently in the reaction mixture containing 20 mM indole, 3M KHCO3, 100mM potassium phosphate buffer (pH 6.0) in a tightly closed reaction vessel. By 6h, 6.81 mM indole-3-carboxylic acid accumulated in the reaction mixture with a molar conversion yield of 34%. Compared to the carhoxylation of pyrrole by pyrrole-2-carboxylate decarboxylase, the lower value compared might derive from the lower solubility of indole in 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... 

Also interesting is the synthesis of pyrrole-2-carboxylate from carbon dioxide and pyrrole using reverse reaction of pyrrole-2-carboxylate decarboxylase enzyme... 

Wieser M, Yoshida T, Nagasawa T (2001) Carbon dioxide fixation by reversible pyrrole-2-carboxylate decarboxylase and its application. J Mol Catal B Enzym 11 179-184... 

Omura H, Wieser M, Nagasawa T (1998) Pyrrole-2-carboxylate decarboxylase from Bacillus megaterium PYR2910, an organic-acid-requtring enzyme. Em J Biochem 253 480-484... 

As the reaction catalyzed by decarboxylase is essentially irreversible, the reverse carbox-ylation reaction is not possible in the practical sense under conventional reaction conditions. An example, however, has made a breakthrough under conditions with very high concentrations of bicarbonate ion in the solution. Pyrrole (82) was carboxylated to give pyrrole-2-carboxylate (83) by the aid of the pyrrole-2-carboxylate decarboxylase of Bacillus megaterium PYR 2910 in a solution containing 3 M KHCO3 [Eq. (28)]. The conversion reached 80%, and the reaction is quite effective, considering its reaction equilibrium [138,139]. 

We have presented evidence that pyrrole-2-carboxylic acid decarboxylates in acid via the addition of water to the carboxyl group, rather than by direct formation of C02.73 This leads to the formation of the conjugate acid of carbonic acid, C(OH)3+, which rapidly dissociates into protonated water and carbon dioxide (Scheme 9). The pKA for protonation of the a-carbon acid of pyrrole is —3.8.74 Although this mechanism of decarboxylation is more complex than the typical dissociative mechanism generating carbon dioxide, the weak carbanion formed will be a poor nucleophile and will not be subject to internal return. However, this leads to a point of interest, in that an enzyme catalyzes the decarboxylation and carboxylation of pyrrole-2-carboxylic acid and pyrrole respectively.75 In the decarboxylation reaction, unlike the case of 2-ketoacids, the enzyme cannot access the potential catalysis available from preventing the internal return from a highly basic carbanion, which could be the reason that the rates of decarboxylation are more comparable to those in solution. Therefore, the enzyme cannot achieve further acceleration of decarboxylation. In the carboxylation of pyrrole, the absence of a reactive carbanion will also make the reaction more difficult however, in this case it occurs more readily than with other aromatic acid decarboxylases. 

Carboxylation of organic molecules using CO2 has received attention as an environmentally benign synthetic method. A decarboxylase, an enzyme from Bacillus megaterium that catalyzes the elimination of CO2 from organic molecules, has been found to also catalyze the reverse CO2 fixation reactions. " The substrate scope of this enzyme, however, is limited to pyrrole, which is carboxylated to form pyrrole-2-carboxylate (Fig. 10.41(a)). For the carboxylations of phenol and catechol, decarboxylases from Clostridium hydroxybenzoicum have been isolated and employed (Fig. 41(b) and(c)). ... 

Employing supercritical CO under high pressure improved the yield of pyrrole-2-carboxylate from pyrrole using a decarboxylase from Bacillus megaterium PYR 2910 by 12-fold (Figure 3.19) [29]. 

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