Has the Bacterial Biphenyl Catabolic Pathway Evolved Primarily To Degrade Biphenyl? The Diphenylmethane Case

In this work, we have compared the ability of Pandoraea pnomenusa B356 and of Burkholderia xenovorans LB400 to metabolize diphenylmethane and benzophenone, two biphenyl analogs in which the phenyl rings are bonded to a single carbon. Both chemicals are of environmental concern. P. pnomenusa B356 grew well on diphenylmethane. On the basis of growth kinetics analyses, diphenylmethane and biphenyl were shown to induce the same catabolic pathway. The profile of metabolites produced during growth of strain B356 on diphenylmethane was the same as the one produced by isolated enzymes of the biphenyl catabolic pathway acting individually or in coupled reactions. The biphenyl dioxygenase oxidizes diphenylmethane to 3-benzylcyclohexa-3,5-diene-1,2-diol very efficiently, and ultimately this metabolite is transformed to phenylacetic acid, which is further metabolized by a lower pathway. Strain B356 was also able to cometabolize benzophenone through its biphenyl pathway, although in this case, this substrate was unable to induce the biphenyl catabolic pathway and the degradation was incomplete, with accumulation of 2-hydroxy-6,7-dioxo-7-phenylheptanoic acid. Unlike strain B356, B. xenovorans LB400 did not grow on diphenylmethane. Its biphenyl pathway enzymes metabolized diphenylmethane, but they poorly metabolize benzophenone. The fact that the biphenyl catabolic pathway of strain B356 metabolized diphenylmethane and benzophenone more efficiently than that of strain LB400 brings us to postulate that in strain B356, this pathway evolved divergently to serve other functions not related to biphenyl degradation.     

The inhibition of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) dehydrochlorinase and gluthathione S-aryltransferase in grass-grub and housefly preparations

The inhibition of DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] dehydrochlorinase and glutathione S-aryltransferase by diphenylmethane and triphenylmethane derivatives was examined. Bis-(3,5-dibromo-4-hydroxyphenyl)methane and similar compounds were excellent inhibitors of both enzymes, but only DDT dehydrochlorinase was inhibited by compounds similar to bis-(N-dimethylaminophenyl)methane. Colour salts of the basic triphenylmethyl dyes were excellent inhibitors of both enzymes. All the inhibitors examined appeared to act by competition with glutathione for its binding site on the two enzymes.     

Influence of Side-Chain Substituents on the Position of Cleavage of the Benzene Ring by Pseudomonas fluorescens

Pseudomonas fluorescens was grown on mineral salts media with phenol, p-hydroxybenzoic acid, p-hydroxy-phenylacetic acid, or p-hydroxy-trans-cinnamic acid as sole carbon and energy source. Each compound was first hydroxylated, ortho to the hydroxyl group on the benzene ring, to give catechol, protocatechuic acid (3,4-dihydroxy-benzoic acid), homoprotocatechuic acid (3,4-dihydroxy-phenylacetic acid), and caffeic acid (3,4-dihydroxy-trans-cinnamic acid), respectively, as the ultimate aromatic products before cleavage of the benzene nucleus. Protocatechuic acid and caffeic acid were shown to be cleaved by ortho fission, via a 3,4-oxygenase mechanism, to give beta-substituted cis, cis-muconic acids as the initial aliphatic products. However, catechol and homoprotocatechuic acid were cleaved by meta fission, by 2,3-and 4,5-oxygenases, respectively, to give alpha-hydroxy-muconic semialdehyde and alpha-hydroxy-gamma-carboxymethyl muconic semialdehyde as initial aliphatic intermediates. Caffeic acid: 3,4-oxygenase, a new oxygenase, consumes 1 mole of O(2) per mole of substrate and has an optimal pH of 7.0. The mechanism of cleavage of enzymes derepressed for substituted catechols by P. fluorescens apparently changes from ortho to meta with the increasing nephelauxetic (electron donor) effect of the side-chain substituent.     

Arhodomonas sp. Strain Seminole and Its Genetic Potential To Degrade Aromatic Compounds under High-Salinity Conditions

Arhodomonas sp. strain Seminole was isolated from a crude oil-impacted brine soil and shown to degrade benzene, toluene, phenol, 4-hydroxybenzoic acid (4-HBA), protocatechuic acid (PCA), and phenylacetic acid (PAA) as the sole sources of carbon at high salinity. Seminole is a member of the genus Arhodomonas in the class Gammaproteobacteria, sharing 96% 16S rRNA gene sequence similarity with Arhodomonas aquaeolei HA-1. Analysis of the genome predicted a number of catabolic genes for the metabolism of benzene, toluene, 4-HBA, and PAA. The predicted pathways were corroborated by identification of enzymes present in the cytosolic proteomes of cells grown on aromatic compounds using liquid chromatography-mass spectrometry. Genome analysis predicted a cluster of 19 genes necessary for the breakdown of benzene or toluene to acetyl coenzyme A (acetyl-CoA) and pyruvate. Of these, 12 enzymes were identified in the proteome of toluene-grown cells compared to lactate-grown cells. Genomic analysis predicted 11 genes required for 4-HBA degradation to form the tricarboxylic acid (TCA) cycle intermediates. Of these, proteomic analysis of 4-HBA-grown cells identified 6 key enzymes involved in the 4-HBA degradation pathway. Similarly, 15 genes needed for the degradation of PAA to the TCA cycle intermediates were predicted. Of these, 9 enzymes of the PAA degradation pathway were identified only in PAA-grown cells and not in lactate-grown cells. Overall, we were able to reconstruct catabolic steps for the breakdown of a variety of aromatic compounds in an extreme halophile, strain Seminole. Such knowledge is important for understanding the role of Arhodomonas spp. in the natural attenuation of hydrocarbon-impacted hypersaline environments.     

Biodegradation of indole at high concentration by persolvent fermentation with Pseudomonas sp. ST-200

Pseudomonas sp. strain ST-200 grew on indole as a sole carbon source. The minimal inhibitory concentration of indole was 0.3 mg/ml for ST-200. However, ST-200 grew in a persolvent fermentation system containing a large amount of indole (a medium containing 20% by vol. diphenylmethane and 4 mg/ml indole), because most of the indole was partitioned in the organic solvent layer. When the organism was grown in the medium containing indole at 1 mg/ml in the presence of diphenylmethane, more than 98% of the indole was consumed after 48 h. Isatic acid (0.4 mg/ml) and isatin (0.03 mg/ml) were produced as the metabolites in the aqueous medium layer. Received: September 12, 1996 / Accepted: January 2, 1997     

Fluorene Oxidation In Vivo by Phanerochaete chrysosporium and In Vitro during Manganese Peroxidase-Dependent Lipid Peroxidation

The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin peroxidase, was studied in liquid cultures of Phanerochaete chrysosporium and in vitro with P. chrysosporium extracellular enzymes. Intact fungal cultures metabolized fluorene to 9-hydroxyfluorene via 9-fluorenone. Some conversion to more-polar products was also observed. Oxidation of fluorene to 9-fluorenone was also obtained in vitro in a system that contained manganese(II), unsaturated fatty acid, and either crude P. chrysosporium peroxidases or purified recombinant manganese peroxidase. The oxidation of fluorene in vitro was inhibited by the free-radical scavenger butylated hydroxytoluene but not by the lignin peroxidase inhibitor NaVO(inf3). Manganese(III)-malonic acid complexes could not oxidize fluorene. These results indicate that fluorene oxidation in vitro was a consequence of lipid peroxidation mediated by P. chrysosporium manganese peroxidase. The rates of fluorene and diphenylmethane disappearance in vitro were significantly faster than those of true polycyclic aromatic hydrocarbons or fluoranthenes, whose rates of disappearance were ionization potential dependent. This result indicates that the initial oxidation of fluorene proceeds by mechanisms other than electron abstraction and that benzylic hydrogen abstraction is probably the route for oxidation.     

Proteogenomic Elucidation of the Initial Steps in the Benzene Degradation Pathway of a Novel Halophile,Arhodomonas sp. Strain Rozel,Isolated from a Hypersaline Environment

Lately, there has been a special interest in understanding the role of halophilic and halotolerant organisms for their ability to degrade hydrocarbons. The focus of this study was to investigate the genes and enzymes involved in the initial steps of the benzene degradation pathway in halophiles. The extremely halophilic bacteria Arhodomonas sp. strain Seminole and Arhodomonas sp. strain Rozel, which degrade benzene and toluene as the sole carbon source at high salinity (0.5 to 4 M NaCl), were isolated from enrichments developed from contaminated hypersaline environments. To obtain insights into the physiology of this novel group of organisms, a draft genome sequence of the Seminole strain was obtained. A cluster of 13 genes predicted to be functional in the hydrocarbon degradation pathway was identified from the sequence. Two-dimensional (2D) gel electrophoresis and liquid chromatography-mass spectrometry were used to corroborate the role of the predicted open reading frames (ORFs). ORFs 1080 and 1082 were identified as components of a multicomponent phenol hydroxylase complex, and ORF 1086 was identified as catechol 2,3-dioxygenase (2,3-CAT). Based on this analysis, it was hypothesized that benzene is converted to phenol and then to catechol by phenol hydroxylase components. The resulting catechol undergoes ring cleavage via the meta pathway by 2,3-CAT to form 2-hydroxymuconic semialdehyde, which enters the tricarboxylic acid cycle. To substantiate these findings, the Rozel strain was grown on deuterated benzene, and gas chromatography-mass spectrometry detected deuterated phenol as the initial intermediate of benzene degradation. These studies establish the initial steps of the benzene degradation pathway in halophiles.     

Application of polyethylene glycol-modified enzymes in biotechnological processes: organic solvent-soluble enzymes

A new approach in biotechnological processes is to use enzymes modified with polyethylene glycol which has both hydrophilic and hydrophobic properties. The modified enzymes are soluble in organic solvents such as benzene, toluene and chlorinated hydrocarbons and exhibit high enzymic activities in these organic solvents. Modified hydrolytic enzymes catalysed the reverse reaction of hydrolysis in organic solvents: formation of acid—amide bonds by modified chymotrypsin, and ester synthesis and ester exchange reactions by modified lipase. Modified catalase and modified peroxidase efficiently catalyse their respective reactions in organic solvents. The results of this research indicate great potential for applications in the fields of biotechnology and enzymology.     

Catabolism of L-tyrosine by the homoprotocatechuate pathway in gram-positive bacteria.

A metabolic pathway for L-tyrosine catabolism involves 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid) as substrate for fission of the benzene nucleus. Cell extracts of an organism tentatively identified as a Micrococcus possessed the enzymes required for degrading homoprotocatechuate to succinate and pyruvate, and stoichiometry was established for several of these reactions. When the required coenzymes were added, cell extracts degraded L-tyrosine to the ring-fission product of homoprotocatechuate 2,3-dioxygenase and also converted 4-hydroxyphenylpyruvic acid into 4-hydroxyphenylacetic acid. This compound, in turn, gave stoichiometric amounts of the ring-fission product of homoprotocatechuate by the action of a nicotinamide adenine dinucleotide phosphate-dependent 3-hydroxylase coupled with homoprotocatechuate 2,3-dioxygenase. Evidence is presented that this route for L-tyrosine catabolism is taken by five other gram-positive strains, including Micrococcus lysodeikticus and a species of Bacillus. Five other gram-positive bacteria from other genera employed the alternative homogentisate pathway.     

A natural isolate of Pseudomonas maltophila which degrades aromatic sulfonic acids

Abstract A natural isolate, designated BSA56, which was originally selected for growth with benzene sulfuric acid as sole carbon and energy source, was identified as a strain of Pseudomonas maltophila . Strain BSA56 grew on a wide range of aromatic sulfonic acids and was shown to release sulfite from benzene sulfonic acid and 2-napthalene sulforic acid. Although it also grew on toluene sulfonic acid and pyridine sulfonic acid, no significant sulfite release was observed with these substrates. Release of sulfite from benzene sulfonic acid was greatly promoted by the presence of glycerol. The ability to release sulfite was induced by growth in the presence of benzene sulfonic acid and was repressed almost entirely by substrates allowing rapid growth such as acetate. Strain BSA56 grew better at 30°C than 37°C on most aromatic substrates, but the reverse was true for most aromatic sulfonates. Several mutants of BSA56 were isolated with defects in benzoate, salicylate, or gentisate metabolism. However, all these mutants retained the ability to degrade the aromatic sulfonates.     

Rapid determination of six urinary benzene metabolites in occupationally exposed and unexposed subjects

A gas chromatography-mass spectrometry method for measurement of the main urinary metabolites of benzene, namely, phenol, catechol, hydroquinone, 1,2,4-trihydroxybenzene (trihydroxybenzene), t,t-muconic acid (muconic acid), and S-phenylmercapturic acid (phenylmercapturic acid), is reported. The method is considerably simpler than existing assays. It was applied to urine from benzene-exposed subjects and controls from Shanghai, China. When subjects were divided into controls (n = 44), those exposed to 31 ppm benzene (n = 19), Spearman correlations with exposure category were >/= 0.728 (p < 0.0001) for all metabolites except trihydroxybenzene. When exposed subjects were compared on an individual basis, all metabolites, including trihydroxybenzene, were significantly correlated with benzene exposure (Pearson r >/= 0.472, p /= 0.708, p < 0.0001). Ratios of individual metabolite levels to total metabolite levels provided evidence of competitive inhibition of CYP 2E1 enzymes leading to increased production of phenol, catechol, and phenylmercapturic acid at the expense of hydroquinone, trihydroxybenzene, and muconic acid. Since all metabolites were detected in all control subjects, the method can be applied to persons exposed to environmental levels of benzene.     

Bacterial Growth Kinetics on Diphenylmethane and Naphthalene-Heptamethylnonane Mixtures

Experiments were carried out to determine if diphenylmethane is utilized by a species of Pseudomonas (Hydrogenomonas) in the dissolved state regardless of the physical state (liquid or solid) of the undissolved diphenylmethane suspended in the medium. Bacterial growth rates in the presence of various amounts of solid or liquid diphenylmethane indicate that liquid diphenylmethane is utilized at the aqueous-diphenylmethane interface but that solid diphenylmethane is not. A Pseudomonas sp. that was isolated on naphthalene (solid), but could not utilize heptamethylnonane, was grown in the presence of various amounts of a naphthalene-heptamethylnonane mixture (liquid). The growth rates indicate that the bacterium could utilize naphthalene at the aqueous-hydrocarbon interface, which is not the case in the absence of the heptamethylnonane.     

Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli

A number of laboratory strains and clinical isolates of Escherichia coli utilized several aromatic acids as sole sources of carbon for growth. E. coli K-12 used separate reactions to convert 3-phenylpropionic and 3-(3-hydroxyphenyl)propionic acids into 3-(2,3-dihydroxyphenyl)propionic acid which, after meta-fission of the benzene nucleus, gave succinate, pyruvate, and acetaldehyde as products. Enzyme assays and respirometry showed that all enzymes of this branched pathway were inducible and that syntheses of enzymes required to convert the two initial growth substrates into 3-(2,3-dihydroxyphenyl)propionate are under separate control. E. coli K-12 also grew with 3-hydroxycinnamic acid as sole source of carbon; the ability of cells to oxidize cinnamic and 3-phenylpropionic acids, and hydroxylated derivatives, was investigated. The lactone of 4-hydroxy-2-ketovaleric acid was isolated from enzymatic reaction mixtures and its properties, including optical activity, were recorded.     

Inhibition of GABA shunt enzymes' activity by 4-hydroxybenzaldehyde derivatives

4-Hydroxybenzaldehyde (HBA) derivatives were examined as inhibitors for GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Investigation of structure-activity relation revealed that a carbonyl group or an amino group as well as a hydroxy group at the para position of the benzene ring are important for both enzymes' inhibition. HBA was shown to give competitive inhibition of GABA-T with respect to alpha-ketoglutarate and competitive inhibition of SSADH. 4-Hydroxybenzylamine (HBM) also showed the competitive inhibition on GABA-T with respect to GABA. In conclusion, the inhibitory effects of HBA and HBM on both enzymes could result from the similarity between both molecules and the two enzymes' substrates in structure, as well as the conjugative effect of the benzene ring. This suggested that the presence of the benzene ring may be accepted by the active site of both enzymes, HBA and HBM may be considered as lead compounds to design novel GABA-T inhibitors.     

Phenylalanine and tyrosine catabolism in some cheese coryneform bacteria

Abstract 23 cheese coryneform bacteria (14 orange, 3 white, and 6 yellow-pigmented) were examined for five enzymes of two branch-point steps in the catabolic pathways of l -phenylalanine and l -tyrosine. Orange cheese coryneforms ( 'Brevibacterium linens' ) catabolized th amino acids by transamination and the benzene ring was cleaved by 3, 4-dihydroxyphenylacetate-2, 3-dioxygenase. Both enzymes appear to be inducible. Yellow and white strains possessed non-inducible low activity of aminotransferase and lacked completely benzene ring cleavage enzymes.     

Isolation and Characterization of Alicycliphilus denitrificans Strain BC, Which Grows on Benzene with Chlorate as the Electron Acceptor

A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 μm wide and 1 to 2 μm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplied.     

Purification and properties of multiple forms of dihydrodiol dehydrogenase from human liver

Two acidic and three basic forms of monomeric dihydrodiol dehydrogenase with molecular weights in the range of 36,000-39,000 were purified from human liver. One acidic enzyme (pI 5.2), which was specific for NADP- and dihydrodiols of benzene and naphthalene, was immunologically identified as aldehyde reductase. The other four enzymes oxidized alicyclic alcohols as well as the dihydrodiols using both NADP+ and NAD+ as cofactors, but showed differences in specificity for hydroxysteroids and inhibitor sensitivity. Two of the basic enzymes (pI 9.7 and 9.1) exhibited a 20 alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas the third basic enzyme (pI 7.6) oxidized some 3 alpha-hydroxysteroids at low rates and was inhibited by cyclopentane-1,1-diacetic acid. Another acidic enzyme, which accounted for the largest amount of enzyme activity in the tissue and appeared in two heterogenous forms with pI values of 5.9 and 5.4, showed a high 3 alpha-hydroxysteroid dehydrogenase activity and was the most sensitive to inhibition by medroxyprogesterone acetate. The Km values of the enzymes, except the pI 5.2 enzyme, for hydroxysteroids (10(-6) to 10(-7) M) were lower than those for xenobiotic alcohols.     

Products Formed from Analogues of 1,1,1-Trichloro-2,2-Bis(p-Chlorophenyl) Ethane (DDT) Metabolites by Pseudomonas putida

Cultures of Pseudomonas putida growing in solutions with diphenylmethane as sole carbon source formed 1,1,1′,1′-tetraphenyldimethyl ether. The product was identified by gas chromatography, mass spectrometry, and infrared and nuclear magnetic resonance spectrometry. The formation of benzophenone, benzhydrol, and phenylglycolic acid was established by gas chromatography and mass spectrometry. Similar techniques also revealed that phenylacetic acid was a major metabolite. Resting cell suspensions converted benzhydrol to phenyl-glycolic acid and products tentatively identified as hydroxybenzhydrols and a hydroxybenzophenone. Cell suspensions of the bacterium also converted the tetraphenyldimethyl ether to benzhydrol and benzophenone. Possible pathways for the degradation of these analogues of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolites are discussed.     

Synthesis of GN8 derivatives and evaluation of their antiprion activity in TSE-infected cells

A series of GN8 derivatives were synthesized from various diamines, carboxylic acid derivatives, and nitrogen nucleophiles, and their antiprion activity was tested in TSE-infected mouse neuronal cells. We found that two ethylenediamine units, hydrophobic substituents on the nitrogen atoms, and the diphenylmethane scaffold were essential structural features responsible for the activity. Seven derivatives bearing substituents at the benzylic position exhibited an improved antiprion activity with the IC₅₀ values of 0.51-0.83 μM. Conformational analysis of model compounds suggested that the introduction of the substituent at the benzylic position restricted the conformational variability of the diphenylmethane unit.