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P W Trudgill

Publications and source records attributed to P W Trudgill.

At least 19 recordsLinked to original sources
PubMed1997

Enzymology of oxidation of tropic acid to phenylacetic acid in metabolism of atropine by Pseudomonas sp. strain AT3.

Pseudomonas sp. strain AT3 grew with dl-tropic acid, the aromatic component of the alkaloid atropine, as the sole source of carbon and energy. Tropic acid-grown cells rapidly oxidized the growth substrate, phenylacetaldehyde, and phenylacetic acid. Crude cell extracts, prepared from dl-tropic acid-grown cells, contained two NAD+-linked dehydrogenases which were separated by ion-exchange chromatography and shown to be specific for their respective substrates, dl-tropic acid and phenylacetaldehyde. Phenylacetaldehyde dehydrogenase was relatively unstable. The stable tropic acid dehydrogenase was purified to homogeneity by a combination of ion-exchange, molecular-sieve, and affinity chromatography. It had a pH optimum of 9.5 and was equally active with both enantiomers of tropic acid, and at this pH, phenylacetaldehyde was the only detectable product of tropic acid oxidation. The formation of phenylacetaldehyde from tropic acid requires, in addition to dehydrogenation, a decarboxylation step. By analogy with NAD+-specific isocitrate and malate dehydrogenases, phenylmalonic semialdehyde, a 3-oxoacid, would be expected to be the precursor of phenylacetaldehyde. Other workers have established that isocitrate and malate dehydrogenases catalyze the decarboxylation of enzyme-bound or added 3-oxoacid intermediates, a reaction that requires Mn2+ or Mg2+ ions. Studies with tropic acid dehydrogenase were hampered by lack of availability of phenylmalonic semialdehyde, but in the absence of added divalent metal ions, both enantiomers of tropic acid were completely oxidized and we have not, by a number of approaches, found any evidence for the transient accumulation of phenylmalonic semialdehyde.

Acetaldehyde↗
PubMed1996

Atropine Metabolism by Pseudomonas sp. Strain AT3: Evidence for Nortropine as an Intermediate in Tropine Breakdown and Reactions Leading to Succinate.

Pseudomonas strain AT3, isolated by elective culture with atropine, hydrolyzed atropine and grew diauxically, first on the tropic acid and then on the tropine. Tropine was also used as a sole carbon and energy source. The methyl group of tropine was eliminated as formaldehyde, and the nortropine thus formed was a precursor of 6-hydroxycyclohepta-1,4-dione. Ammonia was detected as a product of nitrogen elimination. 6-Hydroxycyclohepta-1,4-dione was oxidized to cyclohepta-1,3,5-trione by an induced NAD(sup+)-specific dehydrogenase. Although cyclohepta-1,3,5-trione is a (beta)-diketone with two potential hydrolytic cleavage sites, an induced hydrolase was specific for one of these sites, with 4,6-dioxoheptanoate as the only hydrolysis product. Unlike the alternative cleavage product (3,6-dioxoheptanoate), this compound is also a (beta)-diketone, and a second hydrolytic cleavage formed succinate and acetone. Although Pseudomonas strain AT3 was not capable of growth with acetone, the compound was not detected in the culture medium and may have been lost to the atmosphere. Exhaustive experimentation with a wide range of conditions did not result in detection of the enzymes required for cleavage of the carbon-nitrogen bonds leading to the formation of nortropine and 6-hydroxycyclohepta-1,4-dione.

Journal Article↗
PubMed1995

Tropine dehydrogenase: purification, some properties and an evaluation of its role in the bacterial metabolism of tropine.

Tropine dehydrogenase was induced by growth of Pseudomonas AT3 on atropine, tropine or tropinone. It was NADP(+)-dependent and gave no activity with NAD+. The enzyme was very unstable but a rapid purification procedure using affinity chromatography that gave highly purified enzyme was developed. The enzyme gave a single band on isoelectric focusing with an isoelectric point at approximately pH 4. The native enzyme had an M(r) of 58,000 by gel filtration and 28,000 by SDS/PAGE and therefore consists of two subunits of equal size. The enzyme displayed a narrow range of specificity and was active with tropine and nortropine but not with pseudotropine, pseudonortropine, or a number of related compounds. The apparent Kms were 6.06 microM for tropine and 73.4 microM for nortropine with the specificity constant (Vmax/Km) for tropine 7.8 times that for pseudotropine. The apparent Km for NADP+ was 48 microM. The deuterium of [3-2H]tropine and [3-2H]pseudotropine was retained when these compounds were converted into 6-hydroxycyclohepta-1,4-dione, an intermediate in tropine catabolism, showing that the tropine dehydrogenase, although induced by growth on tropine, is not involved in the catabolic pathway for this compound. 6-Hydroxycyclohepta-1,4-dione was also implicated as an intermediate in the pathways for pseudotropine and tropinone catabolism.

Alcohol Oxidoreductases↗
PubMed1995

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.

Aspergillus fumigatus↗
PubMed1994

4-Ethylphenol metabolism by Aspergillus fumigatus.

Aspergillus fumigatus ATCC 28282 was found to be capable of growth on 4-ethylphenol as its sole carbon and energy source. A pathway for the metabolism of this compound has been proposed. The initial step involves hydroxylation of the methylene group of 4-ethylphenol to form 1-(4'-hydroxyphenyl)ethanol, followed by oxidation to 4-hydroxyacetophenone. The hydroxylase was NADPH and oxygen dependent, which is a characteristic of a monooxygenase type of enzyme. The 1-(4'-hydroxyphenyl)ethanol isolated from growth medium was a racemic mixture of R-(+) and S-(-) enantiomers. 4-Hydroxyacetophenone undergoes an NADPH-dependent Baeyer-Villiger type of oxygenation to give 4-hydroxyphenyl acetate, which is hydrolyzed to form hydroquinone (1,4-dihydroxybenzene). Hydroxylation of hydroquinone by an NADPH-dependent enzyme produces 1,2,4-trihydroxybenzene, the ring fission substrate, which is cleaved by ortho fission to form maleylacetate. The pathway was elucidated by various kinds of investigations. Analysis of culture medium sampled during growth on 4-ethylphenol revealed the transient appearance of 1-(4'-hydroxyphenyl)ethanol, 4-hydroxyacetophenone, and hydroquinone. Cells grown on 4-ethylphenol were able to oxidize all of these compounds immediately, whereas oxidation by succinate-grown cells showed a lag period. Extracts prepared from cells grown on 4-ethylphenol contained enzyme activities for all of the proposed steps. Apart from a low level of esterase activity towards 4-hydroxyphenyl acetate, extracts prepared from cells grown on succinate did not contain any of these enzyme activities.

Acetophenones↗
PubMed1993

The isolation and identification of 6-hydroxycyclohepta-1,4-dione as a novel intermediate in the bacterial degradation of atropine.

Growth of Pseudomonas AT3 on the alkaloid atropine as its sole source of carbon and nitrogen is nitrogen-limited and proceeds by degradation of the tropic acid part of the molecule, with the metabolism of the tropine being limited to the point of release of its nitrogen. A nitrogen-free compound accumulated in the growth medium and was isolated and identified as 6-hydroxycyclohepta-1,4-dione. This novel compound is proposed as an intermediate in tropine metabolism. It served as a growth substrate for the organism and was also the substrate for an NAD(+)-linked dehydrogenase present in cell extracts. The enzyme was induced during the tropine phase of diauxic growth on atropine or during growth on tropine alone.

Alcohol Oxidoreductases↗
PubMed1993

Diketocamphane enantiomer-specific 'Baeyer-Villiger' monooxygenases from camphor-grown Pseudomonas putida ATCC 17453.

Pseudomonas putida ATCC 17453 grew with either (+)- or (-)-camphor as sole carbon source. Enantiomer-specific 'biological Baeyer-Villiger' monooxygenases were synthesized irrespective of the camphor isomer used for growth. The two enzymes are probably the products of separate genes but showed many similarities. Each consisted of two electrophoretically identical subunits, bound flavin mononucleotide (FMN) non-covalently and accepted electrons from an induced NADH dehydrogenase which interacted with the FMN bound to the oxygenating component. They showed minor differences in M(r) with 3,6-diketocamphane 1,6-monooxygenase being the smaller enzyme. Isoelectric focussing showed the two enzymes to have different acidic pI values. Polyclonal antibodies raised against 3,6-diketocamphane 1,6-monooxygenase also cross-reacted with 2,5-diketocamphane 1,2-monooxygenase and its subunits.

Camphor↗
PubMed1993

Metabolism of p-Cresol by the Fungus Aspergillus fumigatus.

The fungus Aspergillus fumigatus ATCC 28282 was shown to grow on p-cresol as its sole source of carbon and energy. A pathway for metabolism of this compound was proposed. This has protocatechuate as the ring-fission substrate with cleavage and metabolism by an ortho-fission pathway. The protocatechuate was formed by two alternative routes, either by initial attack on the methyl group, which is oxidized to carboxyl, followed by ring-hydroxylation, or by ring-hydroxylation as the first step with subsequent oxidation of 4-methylcatechol to the acid. The pathway was elucidated from several pieces of evidence. A number of compounds, including 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, protocatechualdehyde, and 4-methylcatechol, appeared transiently in the medium during growth on p-cresol. These compounds were oxidized without lag by p-cresol-grown cells but not by succinate-grown cells. Enzyme activities for most of the proposed steps were demonstrated in cell extracts after growth on p-cresol, and the products of these activities were identified. None of the activities were found in succinate-grown cells.

Journal Article↗
PubMed1990

Microbial metabolism of monoterpenes--recent developments.

Monoterpenes are important renewable resources for the perfume and flavour industry but the pathways and enzymology of their degradation by microorganisms are not well documented. Until recently the acyclic monoterpene alcohols, (+)-camphor and the isomers of limonene were the only compounds for which significant sections of catabolic pathways and associated enzymology had been reported. In this paper recent developments in our understanding of the enzymology of ring cleavage by microorganisms capable of growth with 1,8-cineole and alpha-pinene are described. 1,8-Cineole has the carbocyclic skeleton of a monocyclic monoterpene with the added complication of an internal ether linkage. Ring hydroxylation strategy and biological Baeyer-Villiger oxygenation lead to an efficient method for cleaving the ether linkage. alpha-Pinene is an unsaturated bicyclic monoterpene hydrocarbon. At least two catabolic pathways exist. Information concerning one of them, in which alpha-pinene may be initially converted into limonene, is rudimentary. The other involves attack at the double bond resulting in formation of alpha-pinene epoxide. Ring cleavage is then catalysed by a novel lyase that requires no additional components and breaks both carbocyclic rings in a concerted manner.

Bicyclic Monoterpenes↗
PubMed1987

Bacterial metabolism of alpha-pinene: pathway from alpha-pinene oxide to acyclic metabolites in Nocardia sp. strain P18.3.

Over 20 gram-positive bacteria were isolated by elective culture with (+/-)-alpha-pinene as the sole carbon source. One of these strains, Nocardia sp. strain P18.3, was selected for detailed study. alpha-Pinene-grown cells oxidized, without lag, alpha-pinene, alpha-pinene oxide (epoxide), and the cis and trans isomers of 2-methyl-5-isopropylhexa-2,5-dienal. No other tested terpene was oxidized at a significant rate. alpha-Pinene was not metabolized by cell extracts in the presence or absence of NADH or NADPH. Cell extracts catalyzed a rapid decyclization of alpha-pinene oxide, in the absence of added cofactors, with the formation of cis-2-methyl-5-isopropylhexa-2,5-dienal. Further oxidation of the aldehyde to the corresponding acid occurred in the presence of NAD. Both activities were induced by growth with alpha-pinene. A rapid, nonenzymic transformation of the cis aldehyde into the trans isomer occurred in glycine buffer. The trans isomer was also a substrate for the NAD-linked aldehyde dehydrogenase. The distribution of the alpha-pinene oxide lyase in alpha-pinene-utilizing Pseudomonas spp. was also investigated and was compatible with the two alternative ring-cleavage sequences that have been proposed on the basis of accumulated metabolites.

Aldehyde-Lyases↗
PubMed1987

Purification and properties of alpha-pinene oxide lyase from Nocardia sp. strain P18.3.

alpha-Pinene oxide is an intermediate in the degradation of alpha-pinene by Nocardia sp. strain P18.3 and some Pseudomonas strains. The epoxide is cleaved by a lyase which catalyzes a concerted reaction in which both rings of the bicyclic structure are cleaved with the formation of cis-2-methyl-5-isopropylhexa-2,5-dienal. The enzyme has been purified to homogeneity from Nocardia sp. strain P18.3. It was induced by growth with alpha-pinene and constituted 6 to 7% of the soluble protein of cell extracts. The apparent molecular weight of the native enzyme was 50,000 by ultracentrifugal analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave two dissimilar subunits with apparent molecular weights of 17,000 and 22,000. The enzyme was devoid of prosthetic groups, had no cofactor requirement, and had a broad pH activity range, a Km for alpha-pinene oxide of 9 microM, and a turnover number of 15,000. Inhibitors included sulfhydryl reactive compounds, terpene epoxides, and pinane derivatives with substituent groups at carbon 3. A mechanism for the concerted reaction has been proposed in which decyclization is initiated by donation of a proton from the catalytic center to the oxygen of the epoxide with consequent destabilization. In vitro the enzyme was inactivated during catalysis, and a reactive cationic intermediate may be responsible for this phenomenon. The enzyme should be classified as a lyase EC 4.99.-.-.

Aldehyde-Lyases↗
PubMed1986

Camphor revisited: studies of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453.

The oxygenating component of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453 was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatography on DEAE-cellulose and polyanion SI-17 columns. It had an Mr of 78,000, bound one molecule of nonautooxidizable flavin mononucleotide (FMN), consisted of two subunits of equal molecular weight, and existed in two electrophoretically distinguishable active forms. The oxygenating complex was constructed from equimolecular amounts of an NADH oxidase, which could be purified separately (Mr, 36,000), and the oxygenating component. Most of the NADH oxidase dissociated from the oxygenating component during purification, although traces remained, to give the final preparation of the oxygenating component significant oxygenase activity. FMN did not dissociate significantly from the oxygenating component during purification, but it was not covalently bound and could be removed under a variety of conditions. Binding between the two proteins that made up the active complex was fairly weak and freely reversible. It probably occurred through the FMN which was strongly bound to the oxygenating component and for which the NADH had a weak binding site. Iron was not present at a significant level in the oxygenating component, and in common with other characterized Baeyer Villiger monooxygenases, 2,5-diketocamphane 1,2-monooxygenase was found to be a simple flavoprotein.

Apoenzymes↗
PubMed1983

Camphor revisited: involvement of a unique monooxygenase in metabolism of 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid by Pseudomonas putida.

Previously, Pseudomonas putida was shown to degrade (+)-camphor, and cleavage of the first ring of the bicyclic structure involved two monooxygenases (a hydroxylase and a ring oxygen-inserting enzyme), a dehydrogenase, and spontaneous cleavage of an unstable oxygenation product (lactone). Cleavage of the second ring was not demonstrated but was assumed also to occur by ring oxygen insertion, since the predicted oxygenation product was extracted from whole-cell incubation systems. Our investigation established that metabolism of the first ring cleavage intermediate, 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid, occurred through the sequential action of two inducible enzymes, a coenzyme A ester synthetase and an oxygenase. The oxygenase was purified to homogeneity and had a molecular weight of 106,000. This enzyme carried a single molecule of flavin adenine dinucleotide and consisted of two identical subunits. Iron was not present at a significant level. The oxygenase was specific for NADPH as the electron donor and absolutely specific for the coenzyme A ester of 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid as the substrate. The reaction stoichiometry was compatible with this enzyme being a monooxygenase, and a mass spectral analysis of the methyl ester of the product confirmed the insertion of a single oxygen atom. The enzyme appeared to be analogous to, although distinct from. 2,5-diketocamphane 1,2-monooxygenase in catalyzing a "biological Baeyer-Villiger" reaction with the formation of a lactone. Structural analogy suggested that this lactone, like the first, was also unstable and susceptible to spontaneous ring opening, although this was not experimentally established.

Camphor↗
PubMed1982

Metabolism of cyclohexaneacetic acid and cyclohexanebutyric acid by Arthrobacter sp. strain CA1.

A strain of Arthrobacter was isolated by enrichment culture with cyclohexaneacetate as the sole source of carbon and grew with a doubling time of 4.2 h. In addition to growing with cyclohexaneacetate, the organism also grew with cyclohexanebutyrate at concentrations not above 0.05%, and with a variety of alicyclic ketones and alcohols. Oxidation of cyclohexaneacetate proceeded through formation of the coenzyme A (CoA) ester followed by initiation of a beta-oxidation cycle. beta-Oxidation was blocked before the second dehydrogenation step due to the formation of a tertiary alcohol, and the side chain was eliminated as acetyl-CoA by the action of (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase. The cyclohexanone thus formed was degraded by a well-described route that involves ring-oxygen insertion by a biological Baeyer-Villiger oxygenase. All enzymes of the proposed metabolic sequence were demonstrated in cell-free extracts. Arthrobacter sp. strain CA1 synthesized constitutive beta-oxidative enzymes, but further induction of enzymes active toward cyclohexaneacetate and its metabolites could occur during growth with the alicyclic acid. Other enzymes of the sequence, (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase and enzymes of cyclohexanone oxidation, were present at negligible levels in succinate-grown cells but induced by growth with cyclohexaneacetate. The oxidation of cyclohexanebutyrate was integrated into the pathway for cyclohexaneacetate oxidation by a single beta-oxidation cycle. Oxidation of the compound could be divided into two phases. Initial oxidation to (1-hydroxycyclohexan-1-yl)acetate could be catalyzed by constitutive enzymes, whereas the further degradation of (1-hydroxycyclohexan-1-yl)acetate was dependent on induced enzyme synthesis which could be inhibited by chloramphenicol with the consequent accumulation of cyclohexaneacetate and (1-hydroxycyclohexan-1-yl)acetate.

Acetates↗