Phenolphosphate carboxylase EC 411 in Thauera aromatica

OH OP03-


Scheme 3

The anaerobic metabolism of phenol in T. aromatica is initiated by the ATP-dependent conversion of phenol to phenylphosphate catalyzed by phenylphosphate synthase (EC 2.7.9.-). The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenolphosphate carboxylase (EC 4.1.1.-).8'29'30 Both enzyme activities are induced in cells grown anoxially on phenol and nitrate and not in cells grown on 4-hydroxybenzoate, the product of this process. Further metabolism of 4-hydroxybenzoate proceeds via benzoyl coenzyme A. Phenylphos-phate synthase and phenylphosphate carboxylase were purified and characterized from T. aromatica.8-31-33

Phenylphosphate synthase consists of three subunits with molecular masses of 70, 40, and 24 kDa. Subunit 1 resembles the central part of classical phospho-enolpyruvate synthase which contains a conserved histidine residue. It catalyzes the exchange of free [14C] phenol and the phenol moiety of phenylphosphate but not the phosphorylation of phenol. Phosphorylation of phenol requires subunit 1, MgATP, and another protein, subunit 2 (40 kDa), which resembles the N-terminal part of phosphoenolpyruvate synthase. Subunit 1 and 2 catalyze the following reaction:

phenol + MgATP + H2O ^ phenylphosphate + MgAMP + orthophosphate

The phosphoryl group in phenylphosphate is derived from the (-phosphate group of ATP. The free energy of ATP hydrolysis obviously favors the trapping of phenol (Km, 0.04 mM), even at a low ambient substrate concentration. The reaction is stimulated several fold by another protein, subunit 3 (24 kDa). The molecular and catalytic features of phenylphosphate synthase resemble those of phospho-enolpyruvate synthase, albeit with interesting modifications.33

Phenylphosphate is converted into 4-hydroxybenzoate by phenylphosphate carboxylase. This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa. Three of the subunits (a 54, (3 53, and 7 10 kDa) were sufficient to catalyze the exchange of 14CO2 and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation. Phenylphosphate carboxylation was restored when the 8 18 kDa subunit was added. As shown in the reaction model of Fig. 3, the 14CO2 exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO2 from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate. Carboxylation of phenylphos-phate requires the addition of 8 subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate. Then a, (, and 7 subunits catalyze the carboxylation of the enzyme-bound energized phenolate intermediate to produce 4-hydroxybenzoate. Phenylphosphate carboxylase acts on phenolic compounds, uses CO2 as a substrate, does not contain biotin or thiamine diphosphate, requires K+ and a divalent metal cation (Mg2+ or Mn2+) for activity, and are strongly inhibited by oxygen.

The purified E. cloacae P240 4-hydroxybenzoate decarboxylase did not catalyze the carboxylation of phenylphosphate,25 indicating phenolphosphate

Hydroxybenzoate Decarboxylase

Phenylphosphate synthase Phenylphosphate carboxylase

Figure 3: Possible reaction mechanism of carboxylation of phenol via phenylphosphate.

Phenylphosphate synthase Phenylphosphate carboxylase

Figure 3: Possible reaction mechanism of carboxylation of phenol via phenylphosphate.

carboxylase (EC 4.1.1.-) was distinguishable from 4-hydroxybenzoate decarboxylases (EC

Based on the anaerobic metabolism of phenol by T. aromatica, Aresta et al. studied the production of 4-hydroxybenzoic acid from the technological aspect.34 35 The crude extract was partially purified by using membranes with a given molecular weight cut-off (MWCO). The enzyme was supported on a solid matrix (agar) to stabilize its activity. The best solid support was revealed to be low-melting agar on which the enzyme had a life of several days to week. In the membrane reactor, as shown in Fig. 4, phenylphosphate was incubated with the enzyme under CO2 atmosphere and the 4-hydroxybenzoic acid accumulated.35 Once all the phosphorylated phenol has been converted, the solution is recovered, and more substrate was added. Membrane was used for the separation of the reaction space where the enzyme is kept, from the space where the reaction product is extracted. The enzyme does not cross the membrane that has a MWCO of 100 kDa. The 4-hydroxybenzoic acid produced in the central reactor, space B, crossed the membrane and was collected in the external part C from where it can be withdrawn with a syringe or downloaded through the stopcock. The lifetime of the enzyme is of several days at room temperature under atmospheric pressure of carbon dioxide that can be used as reaction gas. The process is clean and good results are obtained with a turnover number of approximately 16 000.



Figure 4: Membrane reactor for 4-hydroxybenzoate production using phenolphosphate carboxylase. A membrane (A) separates the reaction space containing the enzyme (B) from water phase where the product is collected (C).

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