Regulation of the biosynthesis of insulin-secretory-granule proteins. Co-ordinate translational control is exerted on some, but not all, granule matrix constituents.

The enzyme 4-ethylphenol methylenehydroxylase was purified from Pseudomonas putida JD1 grown on 4-ethylphenol. It is a flavocytochrome c for which the Mr was found to be 120,000 by ultracentrifuging and 126,000 by gel filtration. The enzyme consists of two flavoprotein subunits each of Mr 50,000 and two cytochrome c subunits each of Mr 10,000. The redox potential of the cytochrome is 240 mV. Hydroxylation proceeds by dehydrogenation and hydration to give 1-(4'-hydroxyphenyl)ethanol, which is also dehydrogenated by the same enzyme to 4-hydroxyacetophenone. The enzyme will hydroxylate p-cresol but is more active with alkylphenols with longer-chain alkyl groups. It is located in the periplasm of the bacterium.     

Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition.

The O2-independent hydroxylase 4-ethylphenol methylenehydroxylase (4EPMH) from Pseudomonas putida JD1 catalysed the complete conversion of 4-ethylphenol into 1-(4-hydroxyphenyl)ethanol together with a small amount of 4-hydroxyacetophenone, but with no formation of the side product 4-vinylphenol reported to be formed when the similar enzyme p-cresol methylhydroxylase (PCMH) catalyses this reaction. The enantiomer of 1-(4-hydroxyphenyl)ethanol produced by 4EPMH was R(+) when horse heart cytochrome c or azurin was used as electron acceptor for the enzyme. PCMHs from various bacterial strains produced the S(-)-alcohol. Both enantiomers of 1-(4-hydroxyphenyl)ethanol were substrates for conversion into 4-hydroxyacetophenone by 4EPMH, but the S(-)-isomer was preferred. The Km and kcat. were 1.2 mM and 41 s-1 respectively for the S(-)-alcohol and 4.7 mM and 22 s-1 for the R(+)-alcohol. In addition to the 1-(4-hydroxyphenyl)ethanol dehydrogenase activity of 4-EPMH, NAD(+)-linked dehydrogenase activity for both enantiomers of the alcohol was found in extracts of Ps. putida JD1.     

4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. A novel flavoprotein catalyzing Baeyer-Villiger oxidation of aromatic compounds.

A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-hydroxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4-hydroxyacetophenone monooxygenase (HAPMO) is a homodimer of approximately 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4-hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer-Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino-acid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of approximately 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer-Villiger monooxygenases: cyclohexanone monooxygenase (27-33% sequence identity) and steroid monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site-directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.     

Regio- and Stereospecific Conversion of 4-Alkylphenols by the Covalent Flavoprotein Vanillyl-Alcohol Oxidase

The regio- and stereospecific conversion of prochiral 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase was investigated. The enzyme was active, with 4-alkylphenols bearing aliphatic side chains of up to seven carbon atoms. Optimal catalytic efficiency occurred with 4-ethylphenol and 4-n-propylphenols. These short-chain 4-alkylphenols are stereoselectively hydroxylated to the corresponding (R)-1-(4′-hydroxyphenyl)alcohols (F. P. Drijfhout, M. W. Fraaije, H. Jongejan, W. J. H. van Berkel, and M. C. R. Franssen, Biotechnol. Bioeng. 59:171–177, 1998). (S)-1-(4′-Hydroxyphenyl)ethanol was found to be a far better substrate than (R)-1-(4′-hydroxyphenyl)ethanol, explaining why during the enzymatic conversion of 4-ethylphenol nearly no 4-hydroxyacetophenone is formed. Medium-chain 4-alkylphenols were exclusively converted by vanillyl-alcohol oxidase to the corresponding 1-(4′-hydroxyphenyl)alkenes. The relative cis-trans stereochemistry of these reactions was strongly dependent on the nature of the alkyl side chain. The enzymatic conversion of 4-sec-butylphenol resulted in two (4′-hydroxyphenyl)-sec-butene isomers with identical masses but different fragmentation patterns. We conclude that the water accessibility of the enzyme active site and the orientation of the hydrophobic alkyl side chain of the substrate are of major importance in determining the regiospecific and stereochemical outcome of vanillyl-alcohol oxidase-mediated conversions of 4-alkylphenols.     

Conversion of 4-hydroxyacetophenone into 4-phenyl acetate by a flavin adenine dinucleotide-containing Baeyer-Villiger-type monooxygenase

An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an M(r) of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed Michaelis-Menten kinetics with apparent K(m) values of 47 microM for 4-hydroxyacetophenone, 384 microM for acetophenone, and 23 microM for 4-hydroxypropiophenone. The apparent K(m) value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 microM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from an Acinetobacter sp.     

Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase

Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum catalyzes the enantioselective hydroxylation of 4-ethylphenol, 4-propylphenol, and 2-methoxy-4-propylphenol into 1-(4'-hydroxyphenyl)ethanol, 1-(4'-hydroxyphenyl)propanol, and 1-(4'-hydroxy-3'-methoxyphenyl)propanol, respectively, with an ee of 94% for the R enantiomer. The stereochemical outcome of the reactions was established by comparing the chiral GC retention times of the products to those of chiral alcohols obtained by the action of the lipases from Candida antarctica and Pseudomonas cepacia. Isotope labeling experiments revealed that the oxygen atom incorporated into the alcoholic products is derived from water. During the VAO-mediated conversion of 4-ethylphenol/4-propylphenol, 4-vinylphenol/4-propenylphenol are formed as side products. With 2-methoxy-4-propylphenol as a substrate, this competing side reaction is nearly abolished, resulting in less than 1% of the vinylic product, isoeugenol. The VAO-mediated conversion of 4-alkylphenols also results in small amounts of phenolic ketones indicative for a consecutive oxidation step. Copyright 1998 John Wiley & Sons, Inc.     

Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium.

A novel bacterium designated strain MV1 was isolated from a sludge enrichment taken from the wastewater treatment plant at a plastics manufacturing facility and shown to degrade 2,2-bis(4-hydroxyphenyl)propane (4,4'-isopropylidenediphenol or bisphenol A). Strain MV1 is a gram-negative, aerobic bacillus that grows on bisphenol A as a sole source of carbon and energy. Total carbon analysis for bisphenol A degradation demonstrated that 60% of the carbon was mineralized to CO2, 20% was associated with the bacterial cells, and 20% was converted to soluble organic compounds. Metabolic intermediates detected in the culture medium during growth on bisphenol A were identified as 4-hydroxybenzoic acid, 4-hydroxyacetophenone, 2,2-bis(4-hydroxyphenyl)-1-propanol, and 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Most of the bisphenol A degraded by strain MV1 is cleaved in some way to form 4-hydroxybenzoic acid and 4-hydroxyacetophenone, which are subsequently mineralized or assimilated into cell carbon. In addition, about 20% of the bisphenol A is hydroxylated to form 2,2-bis(4-hydroxyphenyl)-1-propanol, which is slowly biotransformed to 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Cells that were grown on bisphenol A degraded a variety of bisphenol alkanes, hydroxylated benzoic acids, and hydroxylated acetophenones during resting-cell assays. Transmission electron microscopy of cells grown on bisphenol A revealed lipid storage granules and intracytoplasmic membranes.     

Anaerobic co-metabolic oxidation of 4-alkylphenols with medium-length or long alkyl chains by <Emphasis Type="Italic">Thauera</Emphasis> sp., strain R5

A 4-alkylphenol-degrading facultative anaerobic bacterium, strain R5, was isolated from paddy soil after enrichment with 4-n-propylphenol, 4-n-butylphenol and 4-hydroxybenzoate (4-HBA) under nitrate-reducing conditions. Strain R5 is a Gram-negative rod bacillus grown on phenolic compounds with short alkyl chains (≤C2), organic acids and ethanol. The sequence of the 16S ribosomal RNA gene revealed that the strain is affiliated with Thauera sp. In the presence of 4-HBA as a carbon source, the strain transformed 4-n-alkylphenols with a medium or long-length alkyl chain (C3–C8) to the corresponding oxidised products as follows: 1-(4-hydroxyphenyl)-1-alkenes, -(4-hydroxyphenyl)-1-alkanones and/or 1-(4-hydroxyphenyl)-1-alcohols. The strain also transformed 4-i-propylphenol and 4-sec-butylphenol to (4-hydroxyphenyl)-i-propene and (4-hydroxyphenyl)-sec-butene but not 4-alkylphenols with tertiary alkyl chains (4-t-butylphenol or 4-t-octylphenol). The biotransformation did not proceed without another carbon source and was coupled with nitrate reduction. Biotransformation activity was high in the presence of p-cresol, 4-ethylphenol, 4′-hydroxyacetophenone and 4-HBA as carbon sources and low in the presence of organic acids and ethanol. We suggest that strain R5 co-metabolically transforms alkylphenols to the corresponding metabolites with oxidised alpha carbon in the alkyl chain during coupling with nitrate reduction.     

Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium.

A novel bacterium designated strain MV1 was isolated from a sludge enrichment taken from the wastewater treatment plant at a plastics manufacturing facility and shown to degrade 2,2-bis(4-hydroxyphenyl)propane (4,4'-isopropylidenediphenol or bisphenol A). Strain MV1 is a gram-negative, aerobic bacillus that grows on bisphenol A as a sole source of carbon and energy. Total carbon analysis for bisphenol A degradation demonstrated that 60% of the carbon was mineralized to CO2, 20% was associated with the bacterial cells, and 20% was converted to soluble organic compounds. Metabolic intermediates detected in the culture medium during growth on bisphenol A were identified as 4-hydroxybenzoic acid, 4-hydroxyacetophenone, 2,2-bis(4-hydroxyphenyl)-1-propanol, and 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Most of the bisphenol A degraded by strain MV1 is cleaved in some way to form 4-hydroxybenzoic acid and 4-hydroxyacetophenone, which are subsequently mineralized or assimilated into cell carbon. In addition, about 20% of the bisphenol A is hydroxylated to form 2,2-bis(4-hydroxyphenyl)-1-propanol, which is slowly biotransformed to 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Cells that were grown on bisphenol A degraded a variety of bisphenol alkanes, hydroxylated benzoic acids, and hydroxylated acetophenones during resting-cell assays. Transmission electron microscopy of cells grown on bisphenol A revealed lipid storage granules and intracytoplasmic membranes.     

Anaerobic degradation of p-ethylphenol by "Aromatoleum aromaticum" strain EbN1: pathway, regulation, and involved proteins

The denitrifying “Aromatoleum aromaticum” strain EbN1 was demonstrated to utilize p-ethylphenol under anoxic conditions and was suggested to employ a degradation pathway which is reminiscent of known anaerobic ethylbenzene degradation in the same bacterium: initial hydroxylation of p-ethylphenol to 1-(4-hydroxyphenyl)-ethanol followed by dehydrogenation to p-hydroxyacetophenone. Possibly, subsequent carboxylation and thiolytic cleavage yield p-hydroxybenzoyl-coenzyme A (CoA), which is channeled into the central benzoyl-CoA pathway. Substrate-specific formation of three of the four proposed intermediates was confirmed by gas chromatographic-mass spectrometric analysis and also by applying deuterated p-ethylphenol. Proteins suggested to be involved in this degradation pathway are encoded in a single large operon-like structure (~15 kb). Among them are a p-cresol methylhydroxylase-like protein (PchCF), two predicted alcohol dehydrogenases (ChnA and EbA309), a biotin-dependent carboxylase (XccABC), and a thiolase (TioL). Proteomic analysis (two-dimensional difference gel electrophoresis) revealed their specific and coordinated upregulation in cells adapted to anaerobic growth with p-ethylphenol and p-hydroxyacetophenone (e.g., PchF up to 29-fold). Coregulated proteins of currently unknown function (e.g., EbA329) are possibly involved in p-ethylphenol- and p-hydroxyacetophenone-specific solvent stress responses and related to other aromatic solvent-induced proteins of strain EbN1.     

Elucidation of the 4-hydroxyacetophenone catabolic pathway in Pseudomonas fluorescens ACB

The catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB is known to proceed through the intermediate formation of hydroquinone. Here, we provide evidence that hydroquinone is further degraded through 4-hydroxymuconic semialdehyde and maleylacetate to beta-ketoadipate. The P. fluorescens ACB genes involved in 4-hydroxyacetophenone utilization were cloned and characterized. Sequence analysis of a 15-kb DNA fragment showed the presence of 14 open reading frames containing a gene cluster (hapCDEFGHIBA) of which at least four encoded enzymes are involved in 4-hydroxyacetophenone degradation: 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenyl acetate hydrolase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF). In between hapF and hapB, three genes encoding a putative intradiol dioxygenase (hapG), a protein of the Yci1 family (hapH), and a [2Fe-2S] ferredoxin (hapI) were found. Downstream of the hap genes, five open reading frames are situated encoding three putative regulatory proteins (orf10, orf12, and orf13) and two proteins possibly involved in a membrane efflux pump (orf11 and orf14). Upstream of hapE, two genes (hapC and hapD) were present that showed weak similarity with several iron(II)-dependent extradiol dioxygenases. Based on these findings and additional biochemical evidence, it is proposed that the hapC and hapD gene products are involved in the ring cleavage of hydroquinone.     

Biodegradation of bisphenol A and related compounds by Sphingomonas sp. strain BP-7 isolated from seawater

A bacterium capable of assimilating 2,2-bis(4-hydroxyphenyl)propane (bisphenol A), strain BP-7, was isolated from offshore seawater samples on a medium containing bisphenol A as sole source of carbon and energy, and identified as Sphingomonas sp. strain BP-7. Other strains, Pseudomonas sp. strain BP-14, Pseudomonas sp. strain BP-15, and strain no. 24A, were also isolated from bisphenol A-enrichment culture of the seawater. These strains did not degrade bisphenol A, but accelerated the degradation of bisphenol A by Sphingomonas sp. strain BP-7. A mixed culture of Sphingomonas sp. strain BP-7 and Pseudomonas sp. strain BP-14 showed complete degradation of 100 ppm bisphenol A within 7 d in SSB-YE medium, while Sphingomonas sp. strain BP-7 alone took about 40 d for complete consumption of bisphenol A accompanied by accumulation of 4-hydroxyacetophenone. On a nutritional supplementary medium, Sphingomonas sp. strain BP-7 completely degraded bisphenol A and 4-hydroxyacetophenone within 20 h. The strain degraded a variety of bisphenols, such as 1,1-bis(4-hydroxyphenyl)ethane, 2,2-bis(4-hydroxy-3-methylphenyl)propane, 2,2-bis(4-hydroxyphenyl)butane, and 1,1-bis(4-hydroxyphenyl)cyclohexane, and hydroxy aromatic compounds such as 4-hydroxyacetophenone, 4-hydroxybenzoic acid, catechol, protocatechuic acid, and hydroquinone. The strain did not degrade bis(4-hydroxyphenyl)methane, bis(4-hydroxyphenyl)sulfone, or bis(4-hydroxyphenyl)sulfide.     

19F NMR study on the biological Baeyer-Villiger oxidation of acetophenones

The biological Baeyer–Villiger oxidation of acetophenones was studied by ¹⁹F nuclear magnetic resonance (NMR). The ¹⁹F NMR method was used to characterise the time-dependent conversion of various fluorinated acetophenones in either whole cells of Pseudomonas fluorescens ACB or in incubations with purified 4′-hydroxyacetophenone monooxygenase (HAPMO). Whole cells of P. fluorescens ACB converted 4′-fluoroacetophenone to 4-fluorophenol and 4′-fluoro-2′-hydroxyacetophenone to 4-fluorocatechol without the accumulation of 4′-fluorophenyl acetates. In contrast to 4-fluorophenol, 4-fluorocatechol was further degraded as evidenced by the formation of stoichiometric amounts of fluoride anion. Purified HAPMO catalysed the strictly NADPH-dependent conversion of fluorinated acetophenones to fluorophenyl acetates. Incubations with HAPMO at pH 6 and 8 showed that the enzymatic Baeyer–Villiger oxidation occurred faster at pH 8 but that the phenyl acetates produced were better stabilised at pH 6. Quantum mechanical characteristics explained why 4′-fluoro-2′-hydroxyphenyl acetate was more sensitive to base-catalysed hydrolysis than 4′-fluorophenyl acetate. All together, ¹⁹F NMR proved to be a valid method to evaluate the biological conversion of ring-substituted acetophenones to the corresponding phenyl acetates, which can serve as valuable synthons for further production of industrially relevant chemicals. Journal of Industrial Microbiology & Biotechnology (2001) 26, 35–42. Received 20 April 2000/ Accepted in revised form 16 September 2000     

Biotransformation of biphenyl by Paecilomyces lilacinus and characterization of ring cleavage products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4'-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

The influence of hydroquinone on tyrosinase kinetics

In vitro studies, using combined spectrophotometry and oximetry together with hplc/ms examination of the products of tyrosinase action demonstrate that hydroquinone is not a primary substrate for the enzyme but is vicariously oxidised by a redox exchange mechanism in the presence of either catechol, L-3,4-dihydroxyphenylalanine or 4-ethylphenol. Secondary addition products formed in the presence of hydroquinone are shown to stimulate, rather than inhibit, the kinetics of substrate oxidation.     

Novel Ring Cleavage Products in the Biotransformation of Biphenyl by the Yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4′-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-Trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Stereochemistry of 1-(4''-hydroxyphenyl)ethanol produced by hydroxylation of 4-ethylphenol by p-cresol methylhydroxylase.

Enzymic hydroxylation of 4-ethylphenol by (a) Pseudomonas putida and (b) highly purified p-cresol methylhydroxylase gave optically active 1-(4'-hydroxyphenyl)-ethanol. The products were transformed into the phenolic methyl ethers and shown to contain 69.5% and 65.6%, respectively, of the (S)-(-)-isomer. The stereochemistry of the reaction is discussed in terms of three distinct steps occurring at the active site of the enzyme.     

Hydroquinone dioxygenase from pseudomonas fluorescens ACB: a novel member of the family of nonheme-iron(II)-dependent dioxygenases

Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(II) ions and catalyzed the ring fission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2'-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate (K(i) = 14 microM) was competitive with hydroquinone. Online size-exclusion chromatography-mass spectrometry revealed that HQDO is an alpha2beta2 heterotetramer of 112.4 kDa, which is composed of an alpha-subunit of 17.8 kDa and a beta-subunit of 38.3 kDa. Each beta-subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization--two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases.     

Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.