Biochemical and Molecular Characterization of Phenylacetate-Coenzyme A Ligase, an Enzyme Catalyzing the First Step in Aerobic Metabolism of Phenylacetic Acid in Azoarcus evansii

Abstract

Phenylacetate-coenzyme A ligase (PA-CoA ligase; AMP forming, EC 6.2.1.30), the enzyme catalyzing the first step in the aerobic degradation of phenylacetate (PA) in Azoarcus evansii, has been purified and characterized. The gene (paaK) coding for this enzyme was cloned and sequenced. The enzyme catalyzes the reaction of PA with CoA and MgATP to yield phenylacetyl-CoA (PACoA) plus AMP plus PPi. The enzyme was specifically induced after aerobic growth in a chemically defined medium containing PA or phenylalanine (Phe) as the sole carbon source. Growth with 4-hydroxyphenylacetate, benzoate, adipate, or acetate did not induce the synthesis of this enzyme. This enzymatic activity was detected very early in the exponential phase of growth, and a maximal specific activity of 76 nmol min ؊1 mg of cell protein ؊1 was measured. After 117-fold purification to homogeneity, a specific activity of 48 mol min ؊1 mg of protein Phenylacetic acid and its mono-or dihydroxylated derivatives are formed in nature from a wide variety of natural as well as synthetic compounds. Their microbial biodegradation and biotransformation have received the attention of many research groups for a long time. A number of bacteria and fungi have been isolated and studied to reveal the metabolic pathways of these acids under both aerobic and anaerobic growth conditions. Recently, the complete anaerobic metabolism of phenylacetic acid in the bacterium Thauera aromatica has been shown to follow a novel ␣-oxidation of the side chain of the coenzyme A (CoA)-activated acid, leading to the formation of the central intermediate benzoyl-CoA (Fig. 1). Further metabolism of benzoyl-CoA subsequently leads to the formation of three acetyl-CoA molecules and one CO 2 molecule (21,22,24,31,35,38,39).It has been established that the aerobic metabolism of most aromatic compounds starts by ring hydroxylation reactions carried out by mono-and dioxygenases. These oxic reactions, which are widely distributed in microorganisms, bring the aromatic rings of many aromatic compounds to the redox state present in catechol, protochatechuic and homoprotochatechuic acids, and gentisic and homogentisic acids. These compounds are considered common intermediates in the aerobic metabolism of most aromatic compounds (14,17).The aerobic metabolism of the hydroxylated derivatives of phenylacetic acid in many bacterial species follows this general pattern of oxic attack of the aromatic ring, leading to the formation of homogentisic acid (2,5-dihydroxyphenylactate) or homoprotocatechuic acid (3,4-dihydroxyphenylacetate) (4,5,12,13,28,34,41). However, none of the possible routes which might be expected for phenylacetic acid catabolism by analogy to those of its hydroxy derivatives appears to operate in phenylacetate (PA)-degrading bacteria. The inability to demonstrate the hydroxylation of PA by cell extracts of different bacteria in the presence of different cofactors has been reported by several research groups (7,8,10,15,25,41,43). Therefore, there is still uncertainty about the aerobic ...

Genetic and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2

“…Ibu-2 performs similar reactions with other arylacetic acids, including PAA, 2-phenylpropionic acid, 3-and 4-tolylacetic acids, and 2-(4-tolyl)propionic acid, converting them to the corresponding catechol (or methylcatechol). Although this is somewhat reminiscent of the removal of the carboxyl moiety from benzoate (Eaton, 1996;Fetzner et al, 1992;Jeffrey et al, 1992;Reiner, 1971), it differs from the previously characterized PAA pathways of other bacteria, which do not involve catecholic intermediates (Fernández et al, 2006;Ismail et al, 2003;Martínez-Blanco et al, 1990;El-Said Mohamed, 2000;Rost et al, 2002;Teufel et al, 2010).…”

Metabolic reconstruction of aromatic compounds degradation from the genome of the amazing pollutant-degrading bacteriumCupriavidus necatorJMP134

“…The initial step of the pathway involves the activation of phenylacetate into phenylacetyl‐CoA by a phenylacetate‐coenzyme A ligase, encoded by the paaK gene (Ferrandez et al , 1998). The respective enzymes have also been identified in P. putida (Olivera et al , 1998) and A. evansii (El‐Said Mohamed, 2000). The phenylacetyl‐CoA is attacked by a ring‐oxygenase/reductase (the PaaABCDE gene products), generating a hydroxylated and reduced derivative of phenylacetyl‐CoA, which is not reoxidized to a dihydroxylated aromatic intermediate as in other known aromatic pathways (Fig.…”

3-Hydroxyphenylacetic Acid Induces the Burkholderia cenocepacia Phenylacetic Acid Degradation Pathway ? Toward Understanding the Contribution of Aromatic Catabolism to Pathogenesis

“…The initial step is crucial as it consists of the activation of PA by CoA mediated by the phenylacetate-CoA ligase PaaK (Martinez-Blanco et al, 1990; El-Said Mohamed, 2000). In Burkholderia cenocepacia , the PA degradation genes are organized in three clusters across two of three chromosomes (Figure 1B).…”