David J. Hopper

Evidence of two pathways for the metabolism of phenol by Aspergillus fumigatus

Aspergillus fumigatus (ATCC 28282), a thermotolerant fungus, has been shown to be capable of growth on phenol as the sole carbon and energy source. During growth of the organism on phenol, catechol and hydroquinone accumulated transiently in the medium; cells grown on phenol oxidised these compounds without a lag period. Two different routes operating simultaneously, leading to different ring-fission substrates, are proposed for the metabolism of phenol. In one route, phenol undergoes ortho-hydroxylation to give catechol, which is then cleaved by an intradiol mechanism leading to 3-oxoadipate. In the other route, phenol is hydroxylated in the para-position to produce hydroquinone, which is then converted into 1,2,4-trihydroxybenzene for ring fission by ortho-cleavage to give maleylacetate. Cell-free extracts of phenol-grown mycelia were found to contain enzymic activities for the proposed steps. Two ring-fission dioxygenases, one active towards 1,2,4-trihydroxybenzene, but not catechol, and one active towards both ring-fission substrates, were separated by FPLC. Succinate-grown mycelia did not oxidise any of the intermediates until a clear lag period had elapsed and did not contain any of the enzymic activities for phenol metabolism.

The hydroxylation of P-cresol and its conversion to P-hydroxybenzaldehyde in Pseudomonas putida.

Abstract Cell extracts of Pseudomonas putida catalyze the conversion of p -cresol to p -hydroxybenzylalcohol when phenazine methosulfate, an electron acceptor, is added. The reaction will proceed under anaerobic conditions and a mechanism involving dehydrogenation to a heteroquinone followed by hydration is proposed. This contrasts with the known attack on methyl groups by mono-oxygenases. The same requirements are found for the alcohol dehydrogenase and the major product from reaction mixtures is the aldehyde. Of the compounds tested as substrates only those with the appropriate groups in the para orientation were attacked.

Direct un-mediated electrochemistry of the enzyme P-cresolmethylhydroxylase

The enzyme p-cresolmethylhydroxylase, from Pseudomonas putida, is involved in the degradative pathway of p-cresol. The direct, un-mediated reversible electron transfer between the heme of the flavocytochrome enzyme and an edge-plane graphite electrode in the presence of redox-inactive cationic species as promoters of the enzyme electrochemistry is reported. Diffusion controlled heterogeneous electron transfer is modulated by the levels and type of promoter used, which range from simple and complex cations to polyamines and aminoglycosides. The promoter-enzyme assembly affords a degree of macromolecular recognition at the electrode as both the cationic charge and the flexible nature of the promoter are important to achieve direct electron transfer. A quasi-reversible cyclic voltammetric response was observed in the absence of substrate from which the heterogeneous rate of electron transfer between the enzyme and the electrode was determined as ks~ 1.8 × 10−4 cm2 s−1. An enzyme titration with optimal concentrations of promoter and substrate p-cresol yielded a linear relationship between catalytic current and enzyme concentration up to 1.2 μM. Similarly, relationships were obtained to p-cresol and p-hydroxybenzylalcohol concentrations with fixed amounts of enzyme and promoter, both yielding a linear current vs. substrate response up to 0.5 m M. Furthermore, the magnitude of the catalytic current of p-cresol is greater than that obtained for mediation with the enzymes natural redox partner, azurin, but comparable to those obtained with a ferrocene mediator. Catalytic response was also obtained at a peptide-modified gold electrode in the presence of a promoter, spermine.

Hydroquinone as the Ring-fission Substrate in the Catabolism of 4-Ethylphenol and 4-Hydroxyacetophenone by Pseudomonas putida JD1

SUMMARY: A bacterium capable of growth on 4-ethylphenol was isolated from soil and identified as Pseudomonas putida. Intact cells grown on 4-ethylphenol rapidly oxidized 4-hydroxyaceto-phenone as well as growth substrate and the bacterium was also capable of growth on 4-hydroxyacetophenone. The initial enzymes for 4-ethylphenol catabolism were still present, although at lower activities, in succinate-grown cells which oxidized 4-ethylphenol to 4-hydroxyacetophenone. Extracts of 4-ethylphenol-grown cells oxidized 4-hydroxyacetophenone when provided with NADPH. When this activity was partially purified a stoichiometry of 1 μmol O2 consumed per μmol of substrate was observed with the production of hydroquinone as required for a monooxygenase producing 4-hydroxyphernyl acetate followed by hydrolysis by an esterase. Cell extracts contained esterase activity and hydrolysed 4-hydroxyphenyl acetate to yield hydroquinone. Intact cells converted the analogue, acetophenone, into phenol. Hydroquinone served as the ring-fission substrate and was cleaved by an O2-requiring reaction. The enzymes of the proposed pathway were induced by growth on 4-ethylphenol or 4-hydroxyacetophenone.

The role of His117 in the redox reactions of azurin from Pseudomonas aeruginosa

The electron‐transfer properties of H117G‐ and wild‐type azurin were compared by applying both as electron acceptors in the conversion of 4‐ethylphenol by 4‐ethylphenol methylenehydroxylase (4‐EPMH). The reactivity of H117G‐azurin was determined in the absence and presence of imidazoles, which can substitute the missing fourth ligand. In the absence of imidazoles, H117G‐azurin reacted directly with 4‐ethylphenol, this reaction was abolished in the presence of imidazoles. The enzymatic reduction of H117G‐azurin by 4‐EPMH was 40 times slower than that of wild‐type azurin. The rate of this reaction was enhanced by some imidazoles, diminished by others. In all cases the reduction of H117G‐azurin was irreversible. These results demonstrate that His117 is vital for electron transfer and effectively protects the copper site against aspecific reactions.

Periplasmic location of p-cresol methylhydroxylase in Pseudomonas putida

The cellular location of the flavocytochrome c, p‐cresol methylhydroxylase was investigated in two strains of Pseudomonas putida. In both cases the enzymes were shown to be located in the periplasmic fraction by their release during treatment of the bacteria with EDTA and lysozyme in a solution containing a high concentration of sucrose. For strain NCIB 9869 the finding is in accord with the suggestion that the physiological acceptor for the enyme is azurin as this too was shown to be located mostly in the periplasm.

Alkylphenol Biotransformations Catalyzed by 4-Ethylphenol Methylenehydroxylase

ABSTRACT 4-Ethylphenol methylenehydroxylase from Pseudomonas putida JD1 acts by dehydrogenation of its substrate to give a quinone methide, which is then hydrated to an alcohol. It was shown to be active with a range of 4-alkylphenols as substrates. 4-n-Propylphenol, 4-n-butylphenol, chavicol, and 4-hydroxydiphenylmethane were hydroxylated on the methylene group next to the benzene ring and produced the corresponding chiral alcohol as the major product. The alcohols 1-(4′-hydroxyphenyl)propanol and 1-(4′-hydroxyphenyl)-2-propen-1-ol, produced by the biotransformation of 4-n-propylphenol and chavicol, respectively, were shown to be R(+) enantiomers. 5-Indanol, 6-hydroxytetralin, 4-isopropylphenol, and cyclohexylphenol, with cyclic or branched alkyl groups, gave the corresponding vinyl compounds as their major products.

The catabolism of 4-hydroxyacetophenone by an Alcaligenes sp.

Summary: A bacterium capable of growth on 4-hydroxyacetophenone was isolated from soil and identified as an Alcaligenes sp. Intact cells rapidly oxidized (4-hydroxybenzoyl)methanol, 4-hydroxybenzoate and protocatechuate as well as the growth substrate, and also converted the substrate analogue (4-methoxybenzoyl)methanol to 4-methoxybenzoic acid. When provided with NADH, cell-free extracts oxidized 4-hydroxyacetophenone to 4-hydroxybenzoate and formate, the same products as were formed from (4-hydroxybenzoyl)methanol without NADH. The oxidation of 4-hydroxybenzoate by cell-free extracts required NADPH and the product from both this and protocatechuate oxidation was 3-oxoadipate. A pathway for the catabolism of 4-hydroxyacetophenone, by hydroxylation to (4-hydroxybenzoyl)methanol followed by oxidative cleavage to 4-hydroxybenzoate and formate and hydroxylation of the 4-hydroxybenzoate to protocatechuate, is proposed. Oxidation of protocatechuate was by the ortho pathway. The key enzymes in the proposed pathway were induced by growth on 4-hydroxyacetophenone.

Redox potential of the cytochrome c in the flavocytochrome p-cresol methylhydroxylase

The redox potential of the cytochrome c in 5 flavocytochrome c proteins, all p‐cresol methylhydroxylases purified from species of Pseudomonas, was measured. All gave similar values ranging from 226–250 mV. Two of the enzymes, from Pseudomonas putida NC1B 9866 and NC1B 9869, were resolved into their flavoprotein and cytochrome subunits and the redox potentials of the isolated cytochrome c subunits measured. The values for these were 60–70 mV below those for the whole enzymes but, in both cases, reconstitution of active enzyme by addition of the flavoprotein subunit restored the original potential.

The quinohaemoprotein lupanine hydroxylase from Pseudomonas putida

Lupanine hydroxylase catalyses the first reaction in the catabolism of the alkaloid lupanine by Pseudomonas putida. It dehydrogenates the substrate, which can then be hydrated. It is a monomeric protein of M(r) 72,000 and contains a covalently bound haem and a molecule of PQQ. The gene for this enzyme has been cloned and sequenced and the derived protein sequence has a 26 amino acid signal sequence at the N-terminal for translocation of the protein to the periplasm. Many of the features seen in the sequence of lupanine hydroxylase are common with other quinoproteins including the W-motifs that are characteristic of the eight-bladed propeller structure of methanol dehydrogenase. However, the unusual disulfide bridge between adjacent cysteines that is present in some PQQ-containing enzymes is absent in lupanine hydroxylase. The C-terminal domain contains characteristics of a cytochrome c and overall the sequence shows similarities with that of the quinohaemoprotein, alcohol dehydrogenase from Comamonas testosteroni. The gene coding for lupanine hydroxylase has been successfully expressed in Escherichia coli and a procedure has been developed to renature and reactivate the enzyme, which was found to be associated with the inclusion bodies. Reactivation required addition of PQQ and was dependent on calcium ions.