Pyrrole2carboxylate decarboxylase

Scheme 6

Scheme 6

B. megaterium PYR2910, isolated from soil for its ability to grown on pyrrole-2-carboxylate as the sole source of carbon and energy, produces a novel enzyme, pyrrole-2-carboxylate decarboxylase, to catalyze the decarboxylation of pyrrole-2-carboxylate into pyrrole and CO2.43 Thiophene-2-carboxylate and L-thioproline (each 0.2%, w/v), which are analogous to the substrate pyrrole-2-carboxylate but not converted by the enzyme, were found to be the most effective enzyme inducers, leading to 3-fold higher specific enzyme activity than the substrate inducer pyrrole-2-carboxylate.45

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 Vmax and Km values for decarboxylation were determined to be 989 units mg-1 and 24 mM, respectively. The purified enzyme was stable between pH 6 and 9 and at temperature below 50°C. The pH and temperature optima were 6.5 and 45°C, respectively. A unique feature of this enzyme is its requirement of an organic acid, such as acetate, propionate, butyrate, or pimelate, for the activity. The highest activity was found with pimelate (1750 jxmol min-1 mg-1, Km 1.8 mM), followed by butyrate (76% relative activity, 45 mM), propionate (74%, 42 mM), and acetate (56%, 43 mM). These acids might reflect the steric dimensions of the enzyme site, where the organic acid affects the catalysis. Throughout the reaction, stoichiometric amounts of pyrrole and CO2 were formed from pyrrole-2-carboxylate. During the reaction, the concentrations of added organic acid, such as acetate, stayed constant.43

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 1 h, resulting in an equilibrium constant of 0.3 M.45 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.

o O

100

9

100

80

1 Pyrrole

\ Pyrrole-2-carboxylate

80

\ qOO-o-o

1 ***

60

. tr

Y

60

40

■k

K

40

20

d Pyrrole-2-carboxylate

\ 0 0

20

J

/ Pyrrole

0

i

4 6 8 02468 10 12

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

The reverse CO2 fixation depended on a CO2 source (CO2 or HCO3 -), which was an additional limiting factor for the reverse reaction. Due to high water solubilities, the best CO2 sources were bicarbonates (HCO3 -) with KHCO3 leading to 82 mM pyrrole-2-carboxylate from 100 mM pyrrole, followed by NH4HCO3 (94% relative activity), NaHCO3 (81%), BaCO3 (17%), and CaCO3(16%). Other carbonates (CO32-), CO2 gas, or dry ice were with low or without effect due to a low water solubility of CO32- and CO2 at neutral pH.46 The reverse reaction showed a substrate saturation dependence, with optimal HCO3 - concentrations above 2.5 M.

For the highest carboxylation yield, saturating amounts of 3 M KHCO3 were used, leading to a shift of the reaction equilibrium toward the carboxylate. HCO3 - addition was accompanied by CO2 gas evolution resulting in an increased pressure in the tightly closed reaction vessel of 1.38 atm, which supported the reverse reaction productivity 2.5-fold compared to atmospheric pressure. High pressures are also known to be applied in organic chemical carboxylations.47 As biocatalyst, either concentrated cells with an optical density at 610 nm of 40, previously grown under inducing conditions, or the purified enzyme, both in a concentration corresponding to 100 units enzyme activity ml-1, were employed. Additionally, acetate as an enzyme cofactor and L-ascorbate as an anti-oxidizing and enzyme-protecting agent were added to the reaction mixture. For maximal CO2 fixation rates, 300 mM pyrrole was optimal. Higher pyrrole concentrations inhibited the enzyme. In a batch reaction, 25.5gl-1 (230 mM) pyrrole-2-carboxylate was formed from 20.7gl-1 (300 mM) pyrrole (Fig. 9a). The productivity was increased to 325 mM (36.1gl-1) by feeding 150 mM pyrrole after 3 h, with initially 250 mM pyrrole (Fig. 9b). The yield after bioconversion was 80%, limited by the equilibrium.

Pyrrole-2-carboxylate

02468 10 02468 10 12 Time (min)

Pyrrole-2-carboxylate

02468 10 02468 10 12 Time (min)

Figure 9: Time course of enzymatic carboxylation of pyrrole. (a) In the batch reaction, initially 300 mM pyrrole was added. (b) The batch-fed reaction was started with 250 mM pyrrole, followed by a second addition of 150 mM after 3 h.

An enzyme mechanism including a cofactor role of organic acid was proposed. Analogous to the decarboxylation of pyrrole-2-carboxylate by heat, an electrophilic substitution at C2 pyrrole with a C2 protonated intermediate is probable. The negatively charged organic acid might attack the only positive ring position at N1 attracting its proton. This eases the electron delocalization in the ring, thus increasing the electron density at C2. The C2 proton, therefore, can be substituted by the electrophilic carbon of CO2 (rather than by the less electrophilic carbon of HCO3 -). The electrophilic substitution also allows the reverse decarboxylation with a protonated intermediate stabilized by the organic acid. Support for the presumed catalytic function of the pyrrole Nl-proton might be deduced from the fact that N-methylpyrrole is not converted by the enzyme.

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.48 attempted to carry out the con-

R

Scheme 7

version of pyrrole into pyrrole-2-carboxylate in supercritical CO2 using cells of B. megaterium PYR2910. The reaction was conducted by adding CO2 to 10 MPa to the mixture of pyrrole, the cells, KHCO3, and NH4OAc in potassium phosphate buffer at 40°C. The reaction reached an equilibrium position within a few hours and did not proceed further. The yield of the carboxylation reaction in supercritical CO2 (7.6 MPa) was 12 times higher than that under atmospheric pressure. The effect of pressure on the carboxylation of pyrrole was also investigated, and the maximum yield was between 4 and 7 MPa. This finding suggested the potentiality for biocatalysis in supercritical CO2 in developing synthetic methods utilizing CO2.

Pyrrole-2-carboxylate is employed in the synthesis of various pharmaceuticals4950 and a potential herbicide.51 A number of organic syntheses have been described52-54 However, they require multiple steps and result in low yields. Furthermore, the chemical carbonation of pyrrole with K2CO3 depends on high pressure and temperature.55 The one-step enzymatic conversion has advantages with regard to regiospecificity, yield, and mild reaction conditions.

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