P-cresol Methylhydroxylase Research Articles

Methylhydroxylase encoded by mchAB gene is involved in methyl oxidation of m-cresol via Comamonas thiooxydans CHJ601

The compound m-cresol (meta-cresol) is an important representative of alkylphenol pollutants with high toxicity and potential carcinogenicity. The genes and enzymes associated with the m-cresol pathway have not yet been well elucidated. In this study, Comamonas thiooxydans CHJ601 was isolated from industrial wastewater and cultured utilizing m-cresol as the sole source of carbon and energy. m-Cresol catabolism begins with the oxidation of its methyl group in strain CHJ601. Genome sequencing and heterologous expression of key genes revealed its detailed catabolic pathway. m-Cresol methylhydroxylase (MchAB) catalyzed o-cresol (ortho-cresol), m-cresol, and p-cresol (para-cresol) in the presence of NADH and FAD. The Km value of m-cresol (48.74 ± 5.02 μM) for MchAB was much lower than that of o-cresol (747.80 ± 53.34 μM) and p-cresol (541.90 ± 41.43 μM). Molecular docking analysis showed that the binding affinities of MchAB against m-cresol with two hydrogen bonds are higher than p-cresol methylhydroxylase with one. The optimal substrate of MchAB was m-cresol, which can be catalyzed into 3-hydroxybenzyl alcohol and then oxidized to 3-hydroxybenzaldehyde. The mchC gene encoded 3-hydroxybenzaldehyde dehydrogenase, which catalyzes the oxidation of 3-hydroxybenzaldehyde to 3-hydroxybenzoate. The results indicated that MchAB and MchC are the first characterized enzymes to catalyze the oxidation of the meta-methyl group of m-cresol in the strain CHJ601.

Genetic and physiological characterisation of phenol- and p-cresol-degrading bacteria selected for bioaugmentation in oil- and phenol-polluted area

Successful bioaugmentation requires that bacterial strains introduced into the polluted area must be able to adapt to new environmental conditions and retain high enough catabolic activity. The strains should degrade pollutant present at high concentrations, while having high affinity for the pollutants for their thorough degradation. The transfer of genetic information from introduced donor strain to indigenous bacterial population increases the biodegradation potential. As laboratory-selected strains can be poor survivors and lose catabolic activity in mixed microbial ecosystems, the indigenous biodegradative strains isolated from the river water continuously polluted with phenolic compounds of oil shale leachate may serve as inoculants for bioaugmentation. We have shown that the native phenol- and p-cresol-degrading community could be grouped according to the presence of catabolic genes involved in catabolism of aromatic compounds. The selected representative strains of different catabolic types of degradation of phenol and p-cresol were identified as Pseudomonas mendocina (strain PCl) and P. jluorescens (strains PC! 8, PC24). Catabolic potential of these strains was studied on the basis of phenol hydroxylase, p-cresol methylhydroxylase and catechol 2,3-dioxygenase genes. The occurrence and conjugation of plasmid DNA were revealed in these strains. The ability of the selected strains to degrade several phenolic compounds in natural phenolic wastewater in which the compounds were present in multicomponent mixtures, was investigated through laboratory microcosm studies, To elucidate the extent of interactions among the used bacterial strains single and mixed cultures were set up. The biodegradation activity of strains in microcosms was examined through viable counts, consumption of phenolic compounds and detecting the presence of catabolic genes by hybridization, During the experiments (30 days) the introduced bacteria remained viable even when the substrates were depleted. The mixture of strains was more effective in the decomposition of phenolic compounds from the natural wastewater as compared with the single culture conditions and the metabolic activity and cell density of each strain were co-ordinated within a specific time scale. The behaviour of strains in the phenolic leachate depended on the growth kinetics of the strains (K,,µ).

Mechanism of the Flavoprotein d-6-Hydroxynicotine Oxidase: Substrate Specificity, pH and Solvent Isotope Effects, and Roles of Key Active-Site Residues.

The flavoprotein d-6-hydroxynicotine oxidase catalyzes an early step in the oxidation of ( R)-nicotine, the oxidation of a carbon-nitrogen bond in the pyrrolidine ring of ( R)-6-hydroxynicotine. The enzyme is a member of the vanillyl alcohol oxidase/ p-cresol methylhydroxylase family of flavoproteins. The effects of substrate modifications on the steady-state and rapid-reaction kinetic parameters are not consistent with the quinone-methide mechanism of p-cresol methylhydroxylase. There is no solvent isotope effect on the kcat/ Kamine value with either ( R)-6-hydroxynicotine or the slower substrate ( R)-6-hydroxynornicotine. The effect of pH on the rapid-reaction kinetic parameters establishes that only the neutral form of the substrate and the correctly protonated form of the enzyme bind. The active-site residues Lys348, Glu350, and Glu352 are all properly positioned for substrate binding. The K348M substitution has only a small effect on the kinetic parameters; the E350A and E350Q substitutions decrease the kcat/ Kamine value by ∼20- and ∼220-fold, respectively, and the E352Q substitution decreases this parameter ∼3800-fold. The kcat/ Kamine-pH profile is bell-shaped. The p Ka values in that profile are altered by replacement of ( R)-6-hydroxynicotine with ( R)-6-hydroxynornicotine as the substrate and by the substitutions for Glu350 and Glu352, although the profiles remain bell-shaped. The results are consistent with a network of hydrogen-bonded residues in the active site being involved in binding the neutral form of the amine substrate, followed by the transfer of a hydride from the amine to the flavin.

The catabolism of 2,4-xylenol and p-cresol share the enzymes for the oxidation of para-methyl group in Pseudomonas putida NCIMB 9866

Pseudomonas putida NCIMB 9866 utilizes p-cresol or 2,4-xylenol as a sole carbon and energy source. Enzymes catalyzing the oxidation of the para-methyl group of p-cresol have been studied in detail. However, those responsible for the oxidation of the para-methyl group in 2,4-xylenol catabolism are still not reported. In this study, real-time quantitative PCR analysis indicated pchC- and pchF-encoded p-cresol methylhydroxylase (PCMH) and pchA-encoded p-hydroxybenzaldehyde dehydrogenase (PHBDD) in p-cresol catabolism were also likely involved in the catabolism of 2,4-xylenol. Enzyme activity assays and intermediate identification indicated that the PCMH and PHBDD catalyzed the oxidations of 2,4-xylenol to 4-hydroxy-3-methylbenzaldehyde and 4-hydroxy-3-methylbenzaldehyde to 4-hydroxy-3-methylbenzoic acid, respectively. Furthermore, the PCMH-encoding gene pchF was found to be necessary for the catabolism of 2,4-xylenol, whereas the PHBDD-encoding gene pchA was not essential for the catabolism by gene knockout and complementation. Analyses of the maximum specific growth rate (μ m) and specific activity of the gene-knockout strain to different intermediates revealed the presence of other enzyme(s) with PHBDD activity in strain 9866. However, PHBDD played a major role in the catabolism of 2,4-xylenol in contrast to the other enzyme(s).

Structure and Function of ∆1-Tetrahydrocannabinolic Acid (THCA) Synthase, the Enzyme Controlling the Psychoactivity of Cannabis sativa

∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75Å resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.

Diversity of the transcriptional regulation of the pch gene cluster in two indigenous p-cresol-degradative strains of Pseudomonas fluorescens

p-Cresol methylhydroxylase (PCMH), a key enzyme responsible for the catabolism of p-cresol via the protocatechuate ortho pathway, was used as a tool to characterize catabolic differences between phenol- and p-cresol-degrading Pseudomonas fluore-scens strains PC18 and PC24. Although both strains catabolize p-cresol using PCMH, different whole-cell kinetic parameters for this compound were revealed. Affinity for the substrate and the specific growth rate were higher in PC18, whereas maximum p-cresol tolerance was higher in PC24. In addition, PCMH of strain PC18 was induced during growth on phenol. In both strains, the pchACXF operon, which encodes p-hydroxybenzaldehyde dehydrogenase and PCMH, was sequenced. Transcriptional regulation of these operons by PchR, a putative sigma(54)-dependent regulator, was shown. Although the promoters of these operons resembled sigma(54)-controlled promoters, they differed from the consensus sequence by having T instead of C at position -12. Complementation assays confirmed that the amino acid sequence differences of the PchR regulators between the two strains studied led to different effector-binding capabilities of these proteins: (1) phenol was a more efficient effector for PchR of PC18 than p-cresol, (2) phenol did not activate the regulator of PC24, and (3) both regulators responded similarly to p-cresol.

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8alpha-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8alpha-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.

6-S-Cysteinylation of Bi-covalently Attached FAD in Berberine Bridge Enzyme Tunes the Redox Potential for Optimal Activity

A mutagenic analysis of the amino acid residues His-104 and Cys-166, which are involved in the bi-covalent attachment of FAD to berberine bridge enzyme, was performed. Here we present a detailed biochemical characterization of the cysteine link to FAD observed in this recently discovered group of flavoproteins. The C166A mutant protein still has residual activity, but reduced to approximately 6% of the turnover rate observed for wild-type berberine bridge enzyme. A more detailed analysis of single reaction steps by stopped-flow spectrophotometry showed that the reductive half-reaction is greatly influenced by the lack of the 6-S-cysteinyl linkage, resulting in a 370-fold decrease in the rate of flavin reduction. Determination of the redox potentials for both wild type and the C166A mutein revealed that the difference in the redox potential observed can fully account for the change in the kinetic properties. The wild-type protein exhibits a midpoint potential of +132 mV, which is the highest redox potential determined for any flavoenzyme so far. Removal of the cysteine linkage to FAD in the C166A mutein leads to a redox potential of +53 mV, which is in the expected range for flavoproteins with a single covalent attachment of FAD to a His residue via its 8-alpha position. We also show that the biochemical properties of the mutein resemble that of typical flavoprotein oxidases and that deviations from this behavior observed for the wild type are due to the FAD-6-S-cysteinyl bond. In addition, rapid reaction stopped-flow experiments give no indication for a radical mechanism supporting the direct transfer of a hydride from the substrate to the cofactor.

P-Cresol Methylhydroxylase: Alteration of the Structure of the Flavoprotein Subunit upon Its Binding to the Cytochrome Subunit,

The structures of two forms of a recombinant flavoprotein have been determined at high resolution and compared. These proteins are (1) the flavocytochrome c p-cresol methylhydroxylase (rPCMH, 1.85 A resolution) and (2) the cytochrome-free flavoprotein subunit of rPCMH (PchF, 1.30 A resolution). A significant conformational difference is observed in a protein segment that is in contact with the re face of the isoalloxazine ring of FAD when the structure of PchF is compared to the subunit in the intact flavocytochrome. This structural change is important for optimum catalytic function of the flavoprotein, which has been shown to be dependent on the presence of the cytochrome subunit. This change results in different protein-flavin and apparently different protein-substrate interactions that have a "tuning effect" on the electronic and redox properties of bound p-cresol and the covalently bound FAD. The conformational change in the segment in the cofactor-binding site is induced by a small rearrangement in the flavoprotein-cytochrome interface region of the flavoprotein.

Relationship between Charge-Transfer Interactions, Redox Potentials, and Catalysis for Different Forms of the Flavoprotein Component of p-Cresol Methylhydroxylase

Thirty-three variants of the flavoprotein component of p-cresol methylhydroxylase that contain noncovalently or covalently bound flavin adenine dinucleotide (FAD) analogues were studied. A very good correlation was found between the efficiency of p-cresol oxidation by these proteins and E(CT), the energy for the maximum wavelength for the charge-transfer band of the complex between the bound flavin and 4-bromophenol, a substrate mimic. The correlation covers a range of k(cat) values that spans over 5 orders of magnitude and values of E(CT) that span 900 mV, and the analysis of the data provided a value of the transfer coefficient, alpha, of 0.31. This study demonstrates clearly that the redox properties of both the bound substrate and the flavin cofactor must be taken into account to explain the relative catalytic efficiencies of the variant flavoproteins.

Insight into covalent flavinylation and catalysis from redox, spectral, and kinetic analyses of the R474K mutant of the flavoprotein subunit of p-cresol methylhydroxylase.

Each flavoprotein subunit (PchF) of p-cresol methylhydroxylase (PCMH) has flavin adenine dinucleotide (FAD) covalently tethered to Tyr384. The PCMH structure suggests that Arg474 in PchF is required for self-catalytic covalent flavinylation and for substrate oxidation. The replacement of Arg474 with Lys was carried out to probe the subtleties of the role of Arg474 in these processes. In nearly all of the aspects examined, the mutant protein showed compromised properties relative to the wild-type protein, including the tenacity of noncovalent FAD binding to the apo-protein, the rate of covalent flavinylation, the affinity of the covalent flavoprotein for PchC (the cytochrome subunit), the k(cat) for substrate oxidation, and the affinity for substrate analogues in the formation of FAD-charge-transfer complexes (CT complexes). Nevertheless, because the mutant retains these attributes, the comparison allows for an examination of the role of this residue in the various properties of the enzyme. A correlation is proposed to exist between nu(m), the frequency for the absorbance maximum of the CT complex with a substrate analogue, and k(cat), the steady-state rate constant for oxidation of p-cresol by various forms of PCMH and PchF; both nu(m) and k(cat) can be expressed as functions of the ionization potential of the donor (I(D)) and the electron affinity of the acceptor (E(A)). This correlation is a better predictor of the rate constant for substrate oxidation than is the magnitude of the redox potential, E(m,7), of the bound FAD, which was determined for the various mutant enzyme species and compared with those of the wild type.

Biodegradation of dimethylphenols by bacteria with different ring-cleavage pathways of phenolic compounds.

The biodegradation of 3,4, 2,4, 2,3, 2,6 and 3,5-dimethylphenol in combination with phenol and p-cresol by axenic and mixed cultures of bacteria was investigated. The strains, which degrade phenol and p-cresol through different catabolic pathways, were isolated from river water continuously polluted with phenolic compounds of leachate of oil shale semicoke ash heaps. The proper research of degradation of 2,4 and 3,4-dimethylphenol in multinutrient environments was performed. The degradation of phenolic compounds from mixtures indicated a flux of substrates into different catabolic pathways. Catechol 2,3-dioxygenase activity was induced by dimethylphenols in Pseudomonas mendocina PC1, where meta cleavage pathway was functional during the degradation of p-cresol. In the case of strains PC18 and PC24 of P. fluorescens, the degradation of p-cresol occurred via the protocatechuate ortho pathway and the key enzyme of this pathway, p-cresol methylhydroxylase, was also induced by dimethylphenols. 2,4 and 3,4-dimethylphenols were converted into the dead-end products 4-hydroxy-3-methylbenzoic acid and 4-hydroxy-2-methylbenzoic acid. In the degradation of 3,4-dimethylphenol, the transient accumulation of 4-hydroxy-2-methylbenzaldehyde repressed the consumption of phenol from substrate mixtures. A mixed culture of strains with different catabolic types made it possible to overcome the incompatibilities at degradation of studied substrate mixtures.

Sequence-structure analysis of FAD-containing proteins.

We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein 'pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.

Characterization of the eugenol hydroxylase genes (ehyA/ehyB) from the new eugenol-degrading Pseudomonas sp. strain OPS1.

During the screening for bacteria capable of converting eugenol to vanillin, strain OPS1 was isolated, which was identified as a new Pseudomonas species by 16 s rDNA sequence analysis. When this bacterium was grown on eugenol, the intermediates, coniferyl alcohol, ferulic acid, vanillic acid, and protocatechuic acid, were identified in the culture supernatant. The genes encoding the eugenol hydroxylase (ehyA, ehyB), which catalyzes the first step of this biotransformation, were identified in a genomic library of Pseudomonas sp. strain OPS1 by complementation of the eugenol-negative mutant SK6165 of Pseudomonas sp. strain HR199. EhyA and EhyB exhibited 57% and 85% amino acid identity to the eugenol hydroxylase subunits of Pseudomonas sp. strain HR199 and up to 34% and 54% identity to the corresponding subunits of p-cresol methylhydroxylase from P. putida. Moreover, the amino-terminal sequences of the alpha- and beta-subunits reported recently for an eugenol dehydrogenase of P fluorescens E118 corresponded well with the appropriate regions of EhyA and EhyB. Downstream of ehyB, an open reading frame was identified, whose deduced amino acid sequence exhibited up to 71% identity to azurins, representing most probably the gene (azu) of the physiological electron acceptor of the eugenol hydroxylase. The eugenol hydroxylase genes were amplified by PCR, cloned, and functionally expressed in Escherichia coli.

Reversible accumulation of p-hydroxybenzoate and catechol determines the sequential decomposition of phenolic compounds in mixed substrate cultivations in pseudomonads

Accumulation of key catabolic intermediates during degradation of phenol, p-cresol and benzoate was studied in two-substrate batch cultivations by the strains Pseudomonas mendocina PC1, Pseudomonas fluorescens PC18 and P. fluorescens PC24. According to sequence analysis of 16S rRNA genes the strains belonged to different monophyletic clusters of Pseudomonas. The catechol 2,3-dioxygenase (C23O) gene, xylE, of strain PC1 and the phenol monooxygenase gene, pheA, of PC24 were localised on the chromosome, while the C23O gene, xylE, of strain PC18 and the p-cresol methylhydroxylase gene, pchF, of strains PC18 and PC24 were on plasmids. It was shown that, if the substrates were degraded from mixtures using either catechol meta, catechol ortho or catechol ortho and protocatechuate ortho pathways, then both substrates were catabolised simultaneously (nondiauxic growth) without the accumulation of intermediates. Exceptionally, degradation of phenol and benzoate via the catechol ortho pathway caused irreversible accumulation of cis,cis-muconate without detectable effect on simultaneous consumption of substrates. When the substrates were degraded from mixtures through meta and ortho catabolic pathways, the sequential consumption of substrates (diauxic growth) was observed due to the reversible accumulation of the catabolic intermediates p-hydroxybenzoate or catechol. Regulation of parallel catabolic pathways by the accumulation of catabolic intermediates depended on the concentration of growth substrates. At low concentrations simultaneous degradation occurred and the antagonistic effect of p-hydroxybenzoate on the degradation of phenol was diminished. In strain PC18 only the accumulation of p-hydroxybenzoate during growth on a phenol–p-cresol mixture seems to be directly metabolically regulated because phenol also induces the catabolic pathway for p-cresol degradation. Partial sequencing of the pchF genes of strains PC18 and PC24 showed considerable differences.

Reversible accumulation of p-hydroxybenzoate and catechol determines the sequential decomposition of phenolic compounds in mixed substrate cultivations in pseudomonads

Accumulation of key catabolic intermediates during degradation of phenol, p-cresol and benzoate was studied in two-substrate batch cultivations by the strains Pseudomonas mendocina PC1, Pseudomonas fluorescens PC18 and P. fluorescens PC24. According to sequence analysis of 16S rRNA genes the strains belonged to different monophyletic clusters of Pseudomonas. The catechol 2,3-dioxygenase (C23O) gene, xylE, of strain PC1 and the phenol monooxygenase gene, pheA, of PC24 were localised on the chromosome, while the C23O gene, xylE, of strain PC18 and the p-cresol methylhydroxylase gene, pchF, of strains PC18 and PC24 were on plasmids. It was shown that, if the substrates were degraded from mixtures using either catechol meta, catechol ortho or catechol ortho and protocatechuate ortho pathways, then both substrates were catabolised simultaneously (nondiauxic growth) without the accumulation of intermediates. Exceptionally, degradation of phenol and benzoate via the catechol ortho pathway caused irreversible accumulation of cis, cis-muconate without detectable effect on simultaneous consumption of substrates. When the substrates were degraded from mixtures through meta and ortho catabolic pathways, the sequential consumption of substrates (diauxic growth) was observed due to the reversible accumulation of the catabolic intermediates p-hydroxybenzoate or catechol. Regulation of parallel catabolic pathways by the accumulation of catabolic intermediates depended on the concentration of growth substrates. At low concentrations simultaneous degradation occurred and the antagonistic effect of p-hydroxybenzoate on the degradation of phenol was diminished. In strain PC18 only the accumulation of p-hydroxybenzoate during growth on a phenol– p-cresol mixture seems to be directly metabolically regulated because phenol also induces the catabolic pathway for p-cresol degradation. Partial sequencing of the pchF genes of strains PC18 and PC24 showed considerable differences.

Effects of noncovalent and covalent FAD binding on the redox and catalytic properties of p-cresol methylhydroxylase.

Each flavoprotein subunit (alpha or PchF) of the alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida contains FAD covalently attached to Tyr384. PCMH oxidizes p-cresol to 4-hydroxybenzyl alcohol, which is oxidized subsequently by PCMH to 4-hydroxybenzaldehyde. The Y384F mutant form of PchF (apo-PchF[Y384F]) displayed stoichiometric noncovalent FAD binding. PchF[Y384F]FAD associated with the cytochrome subunit (beta or PchC) (producing PCMH[Y384F]), although not as avidly as with wild-type PchF containing covalently bound FAD (PchF(C)). Dramatic increases in the two-electron E(m,7) (NHE) values for FAD were observed when it bound noncovalently to either apo-PchF or apo-PchF[Y384F], and the two-electron E(m,7) value for FAD was increased further by about 75 mV upon covalent binding to PchF, i.e., PchF(C). The E(m,7) values increased by approximately 20 and 45 mV, respectively, when PchF(C) and PchF[Y384F]FAD associated with PchC. The two-electron E(m,7) for covalently bound FAD in PCMH is 84 mV, the highest measured for a flavoprotein. The values for the one-electron redox potentials (E(m,7), NHE) for FAD were measured also for various forms of PchF. Under anaerobiosis, the reduction of PchF[Y384F]FAD by substrates was similar to that observed previously for PchF containing noncovalently bound FAD. Stopped-flow kinetic studies indicated a rapid substrate reduction of the FAD and heme in PCMH[Y384F] which produced PchF[Y384F]FAD(rad) x PchC, the mutant enzyme containing the flavin radical and reduced heme. These experiments also revealed a slow reduction of unassociated PchC(ox) by PchF[Y384F]FAD(rad) x PchC. Steady-state kinetic studies of the reaction of PCMH[Y384F] with p-cresol indicated that the K(m) for this substrate was unchanged relative to that of PCMH, but that the k(cat) was diminished by an order of magnitude. The data indicate that the covalent attachment of FAD to PchF assists catalysis by raising the E(m,7) of the flavin. Contributions to this effect likely result from conformational changes.

Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of p-cresol methylhydroxylase.

The 9.2-kDa c-type cytochrome subunit (PchC) of the flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida NCIMB 9869 has been overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC, using the recently developed pUCP-Nde vector. Efforts to produce the cytochrome in Escherichia coli using a pET vector, with or without its signal peptide, were generally unsuccessful, yielding relatively low levels of the protein. In contrast, the mature form of PchC accumulated in the periplasmic space of P. aeruginosa PAO1-LAC to about 1 mg/g wet cell paste. A periplasmic fraction enriched to about 12% (w/w) of total protein with recombinant PchC was isolated from the remainder of the cells by a washing procedure using ethylenediaminetetraacetate in the presence of sucrose. The cytochrome was purified to homogeneity from the periplasmic extract by anion-exchange chromatography on DEAE-Sepharose CL-6B followed by chromatofocusing on PolyBuffer Exchanger 94. Purified PchC was obtained in a yield of about 50% and was shown to be identical to that resolved from the native flavocytochrome isolated from P. putida. This system may prove to be of general use for the production of recombinant c-type cytochromes.

Three types of phenol and p-cresol catabolism in phenol- and p-cresol-degrading bacteria isolated from river water continuously polluted with phenolic compounds.

A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species identification by BIOLOG GN analysis revealed 21 strains of Pseudomonas fluorescens (4, 8 and 9 of biotypes A, C and G, respectively), 12 of Pseudomonas mendocina, four of Pseudomonas putida biotype A1, one of Pseudomonas corrugata and one of Acinetobacter genospecies 15. Computer-assisted analysis of rep-PCR fingerprints clustered the strains into groups with good concordance with the BIOLOG GN data. Three main catabolic types of degradation of phenol and p-cresol were revealed. Type I, or meta-meta type (15 strains), was characterized by meta cleavage of catechol by catechol 2,3-dioxygenase (C23O) during the growth on phenol and p-cresol. These strains carried C23O genes which gave PCR products with specific xylE-gene primers. Type II, or ortho-ortho type (13 strains), was characterized by the degradation of phenol through ortho fission of catechol by catechol 1,2-dioxygenase (C12O) and p-cresol via ortho cleavage of protocatechuic acid by protocatechuate 3,4-dioxygenase (PC34O). These strains carried phenol monooxygenase gene which gave PCR products with pheA-gene primers. Type III, or meta-ortho type (11 strains), was characterized by the degradation of phenol by C23O and p-cresol via the protocatechuate ortho pathway by the induction of PC34O and this carried C23O genes which gave PCR products with C23O-gene primers, but not with specific xylE-gene primers. In type III strains phenol also induced the p-cresol protocatechuate pathway, as revealed by the induction of p-cresol methylhydroxylase. These results demonstrate multiplicity of catabolic types of degradation of phenol and p-cresol and the existence of characteristic assemblages of species and specific genotypes among the strains isolated from the polluted river water.

Three types of phenol and p-cresol catabolism in phenol- and p-cresol-degrading bacteria isolated from river water continuously polluted with phenolic compounds

A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species identification by BIOLOG GN analysis revealed 21 strains of Pseudomonas fluorescens (4, 8 and 9 of biotypes A, C and G, respectively), 12 of Pseudomonas mendocina, four of Pseudomonas putida biotype A1, one of Pseudomonas corrugata and one of Acinetobacter genospecies 15. Computer-assisted analysis of rep-PCR fingerprints clustered the strains into groups with good concordance with the BIOLOG GN data. Three main catabolic types of degradation of phenol and p-cresol were revealed. Type I, or meta-meta type (15 strains), was characterized by meta cleavage of catechol by catechol 2,3-dioxygenase (C23O) during the growth on phenol and p-cresol. These strains carried C23O genes which gave PCR products with specific xylE-gene primers. Type II, or ortho-ortho type (13 strains), was characterized by the degradation of phenol through ortho fission of catechol by catechol 1,2-dioxygenase (C12O) and p-cresol via ortho cleavage of protocatechuic acid by protocatechuate 3,4-dioxygenase (PC34O). These strains carried phenol monooxygenase gene which gave PCR products with pheA-gene primers. Type III, or meta-ortho type (11 strains), was characterized by the degradation of phenol by C23O and p-cresol via the protocatechuate ortho pathway by the induction of PC34O and this carried C23O genes which gave PCR products with C23O-gene primers, but not with specific xylE-gene primers. In type III strains phenol also induced the p-cresol protocatechuate pathway, as revealed by the induction of p-cresol methylhydroxylase. These results demonstrate multiplicity of catabolic types of degradation of phenol and p-cresol and the existence of characteristic assemblages of species and specific genotypes among the strains isolated from the polluted river water.