Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1

Abstract

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His(6)PheA1 and His(6)PheA2 were purified and its catalytic activity characterized. His(6)PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His(6)PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His(6)PheA1 and His(6)PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.

Effect of surfactants on the biofilm of Rhodococcus erythropolis, a potent degrader of aromatic pollutants

“…Because of the character of the cell envelope it is able to rapidly colonize various surfaces and form stable biofilm, preferring hydrophobic carriers [39]. The extensive range of organic substances it utilizes was documented also on a broad substrate specificity of the thoroughly studied enzyme phenol hydroxylase [40]. We have already employed this strain in industrial waste water remediation as a biocatalyst in biofilm reactor [41].…”

Catabolism of Phenol and Its Derivatives in Bacteria

“…They consist of a monooxygenase and a flavin reductase subunit. Both subunits, as well as NADH and flavin adenine dinucleotide (FAD), are necessary for phenol hydroxylation (Saa, Jaureguibeitia, Largo, Llama, & Serra, 2010). The flavin reductase subunit catalyzes the NADH-dependent reduction of free FAD.…”

Molecular characterization of a eukaryotic-like phenol hydroxylase from <i>Corynebacterium glutamicum</i>

“…This proposition is supported by the fact that the enzyme activity was significantly decreased upon FAD being lost. Then, the reduced FADH 2 transfers the electron to phenol and reduces it to the corresponding product, catechol (Ohshiro et al, 2004;Saa et al, 2010) (Fig. S2).…”

“…The reaction was started by adding phenol hydroxylase Phe. One enzyme unit is defined as the amount of enzyme which, in the presence of phenol, causes the oxidation of 1 µmol of NADPH per min (Saa et al, 2010). The substrate specificity of Phe was measured by applying different substrates (2 mM) into the phenol hydroxylase enzyme activity reaction system.…”

“…Phenol hydroxylase activity was determined spectrophotometrically at room temperature by measuring the disappearance of NADPH at 340 nm (Neujahr and Gaal, 1973). The assay mixture contained 50 mM phosphate buffer (pH 7.0), 0.15 mM NADPH, 1 mM FAD, 1 mM phenol, 1 mM EDTA (Neujahr and Gaal, 1973;Saa et al, 2010). The bovine serum albumin performed as controls in the reaction system.…”