Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme. Purification and characterization of the reductase moiety.

This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy source, produced a multicomponent enzyme system, located in the cytoplasm and inducible by the substrate, that is responsible for phenol conversion into catechol. Because of the wide diffusion of phenol as a contaminant, the present work represents an initial step towards the biotechnological treatment of waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoretic band. The protein contains a flavin cofactor (FAD) and an iron-sulfur cluster of the type [2Fe-2S]. The function of this reductase is to transfer reducing equivalents from NAD(P)H to the oxygenase component. In vitro, the electron acceptors can be cytochrome c as well as other molecules such as 2, 6-dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazolium. The molecular mass of the reductase was determined to be 41 kDa by SDS/PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N-terminal sequence is similar to those of the reductases from A. calcoaceticus NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductase from A. calcoaceticus BD413. Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase internal peptide sequences but a poorer similarity (3/10) to the internal peptide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413.     

Phenol hydroxylase from Acinetobacter radioresistens S13. Isolation and characterization of the regulatory component.

This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase from Acinetobacter radioresistens S13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron-sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase. PHI is required for catalysis of the conversion of phenol to catechol in vitro, but is not required for PHR activity towards alternative electron acceptors such as cytochrome c and Nitro Blue Tetrazolium. The molecular mass of PHI was determined to be 10 kDa by SDS/PAGE, 8.8 kDa by MALDI-TOF spectrometry and 18 kDa by gel-permeation. This finding suggests that the protein in its native state is a homodimer. The isoelectric point is 4.1. PHI does not contain any redox cofactor and does not bind ANS, a fluorescent probe for hydrophobic sites. The N-terminal sequence is similar to those of the regulatory proteins of phenol hydroxylase from A. calcoaceticus and Pseudomonas CF 600. In the reconstituted system, optimal reaction rate was achieved when the stoichiometry of the components was 2 PHR monomers: 1 PHI dimer: 1 PHO (alphabetagamma) dimer. PHI interacts specifically with PHR, promoting the enhancement of FAD fluorescence emission. This signal is diagnostic of a conformational change of PHR that might result in a better alignment with respect to PHO.     

Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.

Pseudomonas sp. strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmid-encoded phenol hydroxylase and meta-cleavage pathway. The genes encoding the multicomponent phenol hydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment. We report the nucleotide sequence and the polypeptide products of this 5.5-kb region. A combination of deletion analysis, expression of subfragments in tac expression vectors, and identification of polypeptide products in maxicells was used to demonstrate that the polypeptides observed are produced from the six open reading frames identified in the sequence. Expression of phenol hydroxylase activity in a laboratory Pseudomonas strain allows growth on phenol, owing to expression of this enzyme and the chromosomally encoded ortho-cleavage pathway. This system, in conjunction with six plasmids that each expressed all but one of the polypeptides, was used to demonstrate that all six polypeptides are required for growth on phenol.     

PHK from phenol hydroxylase of Pseudomonas sp. OX1. Insight into the role of an accessory protein in bacterial multicomponent monooxygenases

Bacterial multicomponent monooxygenases (BMMs) are members of a wide family of diiron enzymes that use molecular oxygen to hydroxylate a variety of aromatic compounds. The presence of genes encoding for accessory proteins not involved in catalysis and whose role is still elusive, is a common feature of the gene clusters of several BMMs, including phenol hydroxylases and several soluble methane monooxygenases. In this study we have expressed, purified, and partially characterized the accessory component PHK of the phenol hydroxylase from Pseudomonas sp. OX1, a bacterium able to degrade several aromatic compounds. The phenol hydroxylase (ph) gene cluster was expressed in Escherichia coli/JM109 cells in the absence and in the presence of the phk gene. The presence of the phk gene lead to an increase in the hydroxylase activity of whole recombinant cells with phenol. PHK was assessed for its ability to interact with the active hydroxylase complex. Our results show that PHK is neither involved in the catalytic activity of the phenol hydroxylase complex nor required for the assembly of apo-hydroxylase. Our results suggest instead that this component may be responsible for enhancing iron incorporation into the active site of the apo-hydroxylase.     

Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases.

The nucleotide sequences of the Acinetobacter calcoaceticus benABC genes encoding a multicomponent oxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate 1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfer component, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with related sequences, including those for the multicomponent toluate, toluene, benzene, and naphthalene 1,2-dioxygenases, indicated that the similarly sized subunits of the hydroxylase components were derived from a common ancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the alpha-subunits of all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(II) ion. The less conserved beta-subunits of the hydroxylases may be responsible for determining substrate specificity. Each dioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoate dioxygenase, encoded by benC, and the corresponding protein of the toluate 1,2-dioxygenase, encoded by xylZ, were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminal region which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certain monooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfer systems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adenine dinucleotide binding were identified.     

X-ray crystal structure of benzoate 1,2-dioxygenase reductase from Acinetobacter sp. strain ADP1

One of the major processes for aerobic biodegradation of aromatic compounds is initiated by Rieske dioxygenases. Benzoate dioxygenase contains a reductase component, BenC, that is responsible for the two-electron transfer from NADH via FAD and an iron-sulfur cluster to the terminal oxygenase component. Here, we present the structure of BenC from Acinetobacter sp. strain ADP1 at 1.5 A resolution. BenC contains three domains, each binding a redox cofactor: iron-sulfur, FAD and NADH, respectively. The [2Fe-2S] domain is similar to that of plant ferredoxins, and the FAD and NADH domains are similar to members of the ferredoxin:NADPH reductase superfamily. In phthalate dioxygenase reductase, the only other Rieske dioxygenase reductase for which a crystal structure is available, the ferredoxin-like and flavin binding domains are sequentially reversed compared to BenC. The BenC structure shows significant differences in the location of the ferredoxin domain relative to the other domains, compared to phthalate dioxygenase reductase and other known systems containing these three domains. In BenC, the ferredoxin domain interacts with both the flavin and NAD(P)H domains. The iron-sulfur center and the flavin are about 9 A apart, which allows a fast electron transfer. The BenC structure is the first determined for a reductase from the class IB Rieske dioxygenases, whose reductases transfer electrons directly to their oxygenase components. Based on sequence similarities, a very similar structure was modeled for the class III naphthalene dioxygenase reductase, which transfers electrons to an intermediary ferredoxin, rather than the oxygenase component.     

The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13.

Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.     

Iron-based redox centres of reductase and oxygenase components of phenol hydroxylase from A. radioresistens: a redox chain working at highly positive redox potentials

This is the first report of the direct electrochemistry of the reductase (PHR) and oxygenase (PHO) components of phenol hydroxylase from Acinetobacter radioresistens S13 studied by cyclic and differential pulse voltammetry. The PHR contains one 2Fe2S cluster and one FAD that mediate the transfer of electrons from NAD(P)H to the non-heme diiron cluster of PHO. Cyclic and differential pulse voltammetry (CV and DPV) on glassy carbon showed two redox pairs with midpoint potentials at +131.5 ± 13 mV and -234 ± 3 mV versus normal hydrogen electrode (NHE). The first redox couple is attributed to the FeS centre, while the second one corresponds to free FAD released by the protein. DPV scans on native and guanidinium chloride treated PHR highlighted the presence of a split signal (ΔE ≈ 100 mV) attributed to heterogeneous properties of the 2Fe2S cluster interacting with the electrode, possibly due to the presence of two protein conformers and consistently with the large peak-to-peak separation and the peak broadening observed in CV. DPV experiments on gold electrodes performed on PHO confirm a consistently higher reduction potential at +396 mV vs. NHE. The positive redox potentials measured by direct electrochemistry for the FeS cluster in PHR and for the non-heme diiron cluster of PHO show that the entire phenol hydroxylase system works at higher potentials than those reported for structurally similar enzymes, for example methane monooxygenases.     

Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600.

The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pathway. The genes for the entire pathway were previously found to be clustered, and the nucleotide sequences of dmpKLMNOPBC and D, which encode the first four biochemical steps of the pathway, were determined. By using a combination of deletion mapping, nucleotide sequence determinations, and polypeptide analysis, we identified the remaining six genes of the pathway. The fifteen genes, encoded in the order dmpKLMNOPQBCDEFGHI, lie in a single operon structure with intergenic spacing that varies between 0 to 70 nucleotides. Homologies found between the newly determined gene sequences and known genes are reported. Enzyme activity assays of deletion derivatives of the operon expressed in Escherichia coli were used to correlate dmpE, G, H, and I with known meta-cleavage enzymes. Although the function of the dmpQ gene product remains unknown, dmpF was found to encode acetaldehyde dehydrogenase (acylating) activity (acetaldehyde:NAD+ oxidoreductase [coenzyme A acylating]; E.C.1.2.1.10). The role of this previously unknown meta-cleavage pathway enzyme is discussed.     

Nucleotide sequence analysis of genes encoding a toluene/benzene-2-monooxygenase from Pseudomonas sp. strain JS150.

It was previously shown by others that Pseudomonas sp. strain JS150 metabolizes benzene and alkyl- and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, we found that strain JS150 also carries genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen (18O2) incorporation experiments using Pseudomonas aeruginosa strains that carried the cloned genes confirmed that toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Regions encoding the toluene-2-monooxygenase and regulatory gene product were localized in two regions of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined. Analysis of this sequence revealed six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. In addition to these gene products, all the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomonas putida CF600, P35X, and BH than with those from the toluene monooxygenases of Pseudomonas mendocina KR1 and Burkholderia (Pseudomonas) pickettii PKO1. The relationship found between the phenol hydroxylases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested that DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest.     

Cofactor-independent oxygenation reactions catalyzed by soluble methane monooxygenase at the surface of a modified gold electrode.

Soluble methane monooxygenase (sMMO) is a three-component enzyme that catalyses dioxygen- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Oxygenation occurs at the binuclear iron active centre in the hydroxylase component (MMOH), to which electrons are passed from NAD(P)H via the reductase component (MMOR), along a pathway that is facilitated and controlled by the third component, protein B (MMOB). We previously demonstrated that electrons could be passed to MMOH from a hexapeptide-modified gold electrode and thus cyclic voltammetry could be used to measure the redox potentials of the MMOH active site. Here we have shown that the reduction current is enhanced by the presence of catalase or if the reaction is performed in a flow-cell, probably because oxygen is reduced to hydrogen peroxide, by MMOH at the electrode surface and the hydrogen peroxide then inactivates the enzyme unless removed by catalase or a continuous flow of solution. Hydrogen peroxide production appears to be inhibited by MMOB, suggesting that MMOB is controlling the flow of electrons to MMOH as it does in the presence of MMOR and NAD(P)H. Most importantly, in the presence of MMOB and catalase, the electrode-associated MMOH oxygenates acetonitrile to cyanoaldehyde and methane to methanol. Thus the electochemically driven sMMO showed the same catalytic activity and regulation by MMOB as the natural NAD(P)H-driven reaction and may have the potential for development into an economic, NAD(P)H-independent oxygenation catalyst. The significance of the production of hydrogen peroxide, which is not usually observed with the NAD(P)H-driven system, is also discussed.     

Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases

Flavoprotein monooxygenases are involved in a wide variety of biological processes including drug detoxification, biodegradation of aromatic compounds in the environment, biosynthesis of antibiotics and siderophores, and many others. The reactions use NAD(P)H and O2 as co-substrates and insert one atom of oxygen into the substrate. The flavin-dependent monooxygenases utilize a general cycle in which NAD(P)H reduces the flavin, and the reduced flavin reacts with O2 to form a C4a-(hydro)peroxyflavin intermediate, which is the oxygenating agent. This complicated catalytic process has diverse requirements that are difficult to be satisfied by a single site. Two general strategies have evolved to satisfy these requirements. para-Hydroxybenzoate hydroxylase, the paradigm for the single-component flavoprotein monooxygenases, is one of the most thoroughly studied of all enzymes. This enzyme undergoes significant protein and flavin dynamics during catalysis. There is an open conformation that gives access of substrate and product to solvent, and a closed or in conformation for the reaction with oxygen and the hydroxylation to occur. This closed form prevents solvent from destabilizing the hydroperoxyflavin intermediate. Finally, there is an out conformation achieved by movement of the isoalloxazine toward the solvent, which exposes its N5 for hydride delivery from NAD(P)H. The protein coordinates these dynamic events during catalysis. The second strategy uses a reductase to catalyze the reduction of the flavin and an oxygenase that uses the reduced flavin as a substrate to react with oxygen and hydroxylate the organic substrate. These two-component systems must be able to transfer reduced flavin from the reductase to the oxygenase and stabilize a C4a-peroxyflavin until a substrate binds to be hydroxylated, all before flavin oxidation and release of H2O2. Again, protein dynamics are important for catalytic success.     

Rieske business: structure-function of Rieske non-heme oxygenases

Rieske non-heme iron oxygenases (RO) catalyze stereo- and regiospecific reactions. Recently, an explosion of structural information on this class of enzymes has occurred in the literature. ROs are two/three component systems: a reductase component that obtains electrons from NAD(P)H, often a Rieske ferredoxin component that shuttles the electrons and an oxygenase component that performs catalysis. The oxygenase component structures have all shown to be of the alpha3 or alpha3beta3 types. The transfer of electrons happens from the Rieske center to the mononuclear iron of the neighboring subunit via a conserved aspartate, which is shown to be involved in gating electron transport. Molecular oxygen has been shown to bind side-on in naphthalene dioxygenase and a concerted mechanism of oxygen activation and hydroxylation of the ring has been proposed. The orientation of binding of the substrate to the enzyme is hypothesized to control the substrate selectivity and regio-specificity of product formation.     

Benzene dioxygenase in Pseudomonas putida. Subunit composition and immuno-cross-reactivity with other aromatic dioxygenases.

The terminal oxygenase component of benzene dioxygenase from Pseudomonas putida strain ML2 was shown to contain two subunits, of Mr 54,500 and 23,500, by SDS/polyacrylamide-gel electrophoresis. The native Mr of the terminal oxygenase was estimated to be 168,000 +/- 4000. Polyclonal antibodies raised against each of the subunits cross-reacted with two polypeptides in cell-free extracts from toluene-grown Pseudomonas putida strain N.C.I.B. 11767. The Mr values of these polypeptides were similar to those reported for the subunits from the terminal dioxygenase component of toluene dioxygenase. These polypeptides were present only when this strain was grown on toluene. No cross-reactivity was observed with subunits of the naphthalene dioxygenase or benzoate dioxygenase systems.     

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes

Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.     

The multiple nicotinamide nucleotide-binding subunits of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P-labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18-20 kDa. The 51 and the 30-kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [beta-32P]ADP. The 42-kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [beta-32P]ADP. It was not labeled with [32P]NADP(H). The 39-kDa polypeptide was labeled with [32P]NADPH and to a small extent with [beta-32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem. 273, 8094-8098]. The 18-20-kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51-kDa subunit is known to contain FMN and a [4Fe-4S] cluster, and is the NAD(P)H-binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide-binding subunits of complex I have been discussed.     

Phenol Hydroxylase and Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1: Interplay between Two Enzymes

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.     

Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.     

Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes

Eighteen 4-t-octylphenol-degrading bacteria were isolated and screened for the presence of degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from multicomponent phenol hydroxylase, single component monooxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Seventeen of the 18 isolates exhibited the presence of a 232 bp amplicon that shared 61-92% identity to known multicomponent phenol hydroxylase gene sequences from short and/or medium-chain alkylphenol-degrading strains. Twelve of the 18 isolates were positive for a 324 bp region that exhibited 78-95% identity to the closest published catechol 1,2-dioxygenase gene sequences. The two strains, Pseudomonas putida TX2 and Pseudomonas sp. TX1, contained catechol 1,2-dioxygenase genes also have catechol 2,3-dioxygenase genes. Our result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.     

Molecular analysis of a plasmid-encoded phenol hydroxylase from Pseudomonas CF600

Pseudomonas strain CF600 is able to utilize phenol and 3,4-dimethylphenol as sole carbon and energy source. We demonstrate that growth on these substrates is by virtue of plasmid-encoded phenol hydroxylase and a meta-cleavage pathway. Screening of a genomic bank, with DNA from the previously cloned catechol 2,3-dioxygenase gene of the TOL plasmid pWW0, was used in the identification of a clone which could complement a phenol-hydroxylase-deficient transposon insertion mutant. Deletion mapping and polypeptide production analysis identified a 1.2 kb region of DNA encoding a 39.5 kDa polypeptide which mediated this complementation. Enzyme activities and growth properties of Pseudomonas strains harbouring this fragment on a broad-host-range expression vector indicate that phenol hydroxylase is a multicomponent enzyme containing the 39.5 kDa polypeptide as one component.