Phenylphosphate Carboxylase: a New C-C Lyase Involved in Anaerobic Phenol Metabolism in Thauera aromatica

Abstract

The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via carboxylation to 4-hydroxybenzoate and is initiated by the ATP-dependent conversion of phenol to phenylphosphate. The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenylphosphate carboxylase, which was purified and studied. This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa, whose genes are located adjacent to each other in the phenol gene cluster which codes for phenol-induced proteins. Three of the subunits (54, 53, and 10 kDa) were sufficient to catalyze the exchange of 14 CO 2 and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation. Phenylphosphate carboxylation was restored when the 18-kDa subunit was added. The following reaction model is proposed. The 14 CO 2 exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO 2 from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate. Carboxylation of phenylphosphate requires in addition the 18-kDa subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate. The 54-and 53-kDa subunits show similarity to UbiD of Escherichia coli, which catalyzes the decarboxylation of a 4-hydroxybenzoate derivative in ubiquinone (ubi) biosynthesis. They also show similarity to components of various decarboxylases acting on aromatic carboxylic acids, such as 4-hydroxybenzoate or vanillate, whereas the 10-kDa subunit is unique. The 18-kDa subunit belongs to a hydratase/ phosphatase protein family. Phenylphosphate carboxylase is a member of a new family of carboxylases/ decarboxylases that act on phenolic compounds, use CO 2 as a substrate, do not contain biotin or thiamine diphosphate, require K ؉ and a divalent metal cation (Mg 2؉ or Mn 2؉ ) for activity, and are strongly inhibited by oxygen.The anaerobic metabolism of phenol has been studied to some extent in the beta-proteobacterium Thauera aromatica. The two initial steps of the pathway consist of the phosphorylation of phenol to phenylphosphate and the carboxylation of phenylphosphate to 4-hydroxybenzoate (36-38) (Fig. 1). Both enzyme activities are induced in cells grown anoxically on phenol and nitrate and not in cells grown on 4-hydroxybenzoate, the product of this process. 4-Hydroxybenzoate is also an intermediate in the metabolism of p-cresol (53)Further metabolism of 4-hydroxybenzoate proceeds via benzoyl coenzyme A (benzoyl-CoA) in two steps (Fig. 1). A specific CoA ligase forms 4-hydroxybenzoyl-CoA (7, 25), which is reductively dehydroxylated to benzoyl-CoA by a molybdoflavo-iron-sulfur protein, 4-hydroxybenzoyl-CoA reductase (13,15,26). The electron donor is a 2-[4Fe/4S] ferredoxin which is reduced by 2-oxoglutarate-ferredoxin oxidoreductase (21). Benzoyl-CoA is a common intermediate in the metabolism of many aromatic compounds. It is reductively d...

Carboxylases in Natural and Synthetic Microbial Pathways

“…This is exemplified best by acetyl-CoA/propionyl-CoA carboxylase, which is the key enzyme in the autotrophic CO 2 fixation pathways of many archaea, whereas in most bacteria, homologs of this enzyme serve mainly in assimilation and/or biosynthesis. Even more notably, in the case of (21,42,70,106). Thus, it can be expected that even more such enzymes will be discovered.…”

“…Such carboxylations are an important alternative to oxygen-dependent functionalization reactions and are widely used under anoxic conditions. In anaerobic phenol assimilation of Thauera aromatica, phenylphosphate is carboxylated into 4-hydroxybenzoate by phenylphosphate carboxylase (106). Anaerobic metabolism of acetophenone in Aromatoleum aromaticum requires acetophenone carboxylase, which yields benzoylacetate (70).…”

“…Functionalization of inert substrates. The introduction of a carboxylic group is a common strategy in the assimilation of substrates that lack any functionalizable terminal carbon group, such as phenol, acetophenone, epoxypropane, or acetone (39,55,70,106) (Fig. 3).…”

Anaerobic Metabolism of Catechol by the Denitrifying Bacterium Thauera aromatica —a Result of Promiscuous Enzymes and Regulators?

“…Carboxylation of catechol would similarly require catechylphosphate synthase activity (forming catechylphosphate) and catechylphosphate carboxylase (forming 3,4-dihydroxybenzoate [protocatechuate]). The presence of the carboxylase was examined following the carboxylation of phenylphosphate, as well as the exchange of 14 CO 2 into the carboxyl group of 4-hydroxybenzoate and protocatechuate (32). Catechol-and phenol-grown cells contained similar carboxylase activities.…”

“…In brief, concentrated cell suspensions (optical density, 30) were incubated with [ 14 C]phenol or [ 14 C]catechol, and the formation of [ 14 C]phenylphosphate or [ 14 C]catechylphosphate was analyzed by thin-layer chromatography and phosphorimaging. Phenylphosphate carboxylase activity in cell extracts was measured as described previously (32). Phenol-grown cells were used as a positive control.…”