Kegg Pathway: Benzoate degradation via hydroxylation

Org Biomol Chem. 2009 Jun 21; 7(12): 2619-27
Fabris F, Collins J, Sullivan B, Leisch H, Hudlicky T

A series of Benzoate esters (methyl, ethyl, n-Pr, i-Pr, n-Bu, t-Bu, allyl, and propargyl) were subjected to enzymatic dihydroxylation by E. coli JM 109(pDTG 601) strain in a whole-cell fermentation. The cis-cyclohexadienediols were obtained in yields of approximately 1g/L except for n-propyl- and i-propyl Benzoate which were found to be poor substrates. n-Butyl and t-butyl Benzoates were not oxidized at all. The absolute stereochemistry for all metabolites was determined by comparison with a standard prepared from (1S-cis)-3-bromo-3,5-cyclohexadiene-1,2-diol, whose absolute configuration is well established. The free diols were found to be quite stable compared to other cis-dihydrodiols of this type, however, their acetonides underwent a dimerization via a regio- and stereoselective Diels-Alder cycloaddition. The diol derived from ethyl Benzoate was subjected to a stereo- and regioselective inverse electron demand Diels-Alder cycloadditions with several dienophiles. The new adducts were completely characterized. The hetero-Diels-Alder reaction of this diol with an acyl nitroso dienophile yielded regio- and stereoselectively a bicyclic oxazine, which upon reduction provided a useful derivative of amino shikimate that can be exploited in an approach to oseltamivir (Tamiflu) and other amino cyclitols. The diol was also converted to carba-alpha-L-galactopyranose to demonstrate its potential utility as a source of pseudo sugars. Experimental and spectral data are provided for all new compounds.

Org Biomol Chem. 2008 Apr 7; 6(7): 1251-9
Boyd DR, Sharma ND, Harrison JS, Malone JF, McRoberts WC, Hamilton JT, Harper DB

A series of twelve Benzoate esters was metabolised, by species of the Phellinus genus of wood-rotting fungi, to yield the corresponding benzyl alcohol derivatives and eight salicylates. The isolation of a stable oxepine metabolite, from methyl Benzoate, allied to evidence of the migration and retention of a carbomethoxy group (the NIH Shift), during enzyme-catalysed ortho-hydroxylation of alkyl Benzoates to form salicylates, is consistent with a mechanism involving an initial arene epoxidation step. This mechanism was confirmed by the isolation of a remarkably stable, optically active, substituted benzene oxide metabolite of methyl 2-(trifluoromethyl)Benzoate, which slowly converted into the racemic form. The arene oxide was found to undergo a cycloaddition reaction with 4-phenyl-1,2,4-triazoline-3,5-dione to yield a crystalline cycloadduct whose structure and racemic nature was established by X-ray crystallography. The metabolite was also found to undergo some novel benzene oxide reactions, including epoxidation to give an anti-diepoxide, base-catalysed hydrolysis to form a trans-dihydrodiol and acid-catalysed aromatisation to yield a salicylate derivative via the NIH Shift of a carbomethoxy group.

J Org Chem. 2007 Aug 3; 72(16): 6280-3
Lebrasseur N, Gagnepain J, Ozanne-Beaudenon A, Léger JM, Quideau S

SIBX, the nonexplosive formulation of the lambda5-iodane 2-iodoxybenzioc acid (IBX), safely and efficiently mediates the hydroxylative dearomatization of various 2-alkylphenols and napthols into orthoquinols or their [4 + 2] cyclodimers. Reactions are typically run at room temperature using SIBX as a suspension in THF. Using these conditions, natural products such as the cyclodimer of the terpene carvacrol and, for the first time, the shikimate-derived (+/-)-grandifloracin were prepared in one step from their respective phenolic precursor.

Spectrochim Acta A Mol Biomol Spectrosc. 2008 Feb; 69(2): 449-59
Dabbagh HA, Teimouri A, Chermahini AN, Shahraki M

We present a detailed analysis of the structural, infrared spectra and visible spectra of a series of azo dyes preparation of salicylic acid and 2,4-dihydroxy benzoic acid derivatives as the coupling component. The preparation of these azo dyes with salicylic acid and 2,4-dihydroxy benzoic acid derivatives (salicylic acid, methyl salicylate, ethyl salicylate, butyl salicylate, methyl 2,4-dihydroxy Benzoate, ethyl 2,4-dihydroxy Benzoate, salicylaldehyde, salicylamide, 2,4-dihydroxy benzamide, salicylaldoxime) have been investigated theoretically by performing HF and DFT levels of theory using the standard 6-31G* basis set. The optimized geometries and calculated vibrational frequencies are evaluated via comparison with experimental values. The vibrational spectral data obtained from solid phase FT-IR spectra are assigned modes based on the results of the theoretical calculations. The observed spectra are found to be in good agreement with the calculations.

Clin Cancer Res. 2004 Nov 1; 10(21): 7365-74
Chaston TB, Watts RN, Yuan J, Richardson DR

PURPOSE: The development of novel and potent iron chelators as clinically useful antitumor agents is an area of active interest. Antiproliferative activity of chelators often relates to iron deprivation or stimulation of iron-dependent free radical damage. Recently, we showed that novel iron chelators of the di-2-pyridylketone isonicotinoyl hydrazone (PKIH) class have potent and selective antineoplastic activity (E. Becker, et al., Br. J. Pharmacol., 138: 819-30, 2003). In this study, we assessed the effects of the PKIH analogues on the redox activity of iron in terms of understanding their antitumor activity. EXPERIMENTAL DESIGN: We tested the PKIH analogues for their ability to promote iron-mediated ascorbate oxidation, Benzoate hydroxylation, and plasmid degradation. Subsequent experiments assessed their ability to bind DNA, inhibit topoisomerase I, and cause DNA damage. To measure intracellular reactive oxygen species, we used the redox-sensitive probe, 2',7'-dichloro-fluorescein-diacetate, to measure intracellular PKIH-dependent redox activity. RESULTS: The PKIH analogues had relatively little effect on ascorbate oxidation in the presence of Fe(III) but stimulated Benzoate hydroxylation and plasmid DNA degradation in the presence of Fe(II) and H2O2. These ligands could not inhibit DNA topoisomerase I or cause DNA damage in intact cells. PKIH markedly increased the intracellular generation of reactive oxygen species, and this was inhibited by catalase. This enzyme also decreased the antiproliferative effect of PKIH, indicating H2O2 played a role in its cytotoxic activity. CONCLUSIONS: Our results suggest that the antiproliferative effects of these chelators relates to intracellular iron chelation, followed by the stimulation of iron-mediated free radical generation via the so-formed iron complex.

Appl Environ Microbiol. 2004 Aug; 70(8): 4961-70
Denef VJ, Park J, Tsoi TV, Rouillard JM, Zhang H, Wibbenmeyer JA, Verstraete W, Gulari E, Hashsham SA, Tiedje JM

We designed and successfully implemented the use of in situ-synthesized 45-mer oligonucleotide DNA microarrays (XeoChips) for genome-wide expression profiling of Burkholderia xenovorans LB400, which is among the best aerobic polychlorinated biphenyl degraders known so far. We conducted differential gene expression profiling during exponential growth on succinate, Benzoate, and biphenyl as sole carbon sources and investigated the transcriptome of early-stationary-phase cells grown on biphenyl. Based on these experiments, we outlined metabolic pathways and summarized other cellular functions in the organism relevant for biphenyl and Benzoate degradation. All genes previously identified as being directly involved in biphenyl degradation were up-regulated when cells were grown on biphenyl compared to expression in succinate-grown cells. For Benzoate degradation, however, genes for an aerobic coenzyme A activation pathway were up-regulated in biphenyl-grown cells, while the pathway for Benzoate degradation via hydroxylation was up-regulated in Benzoate-grown cells. The early-stationary-phase biphenyl-grown cells showed similar expression of biphenyl pathway genes, but a surprising up-regulation of C(1) metabolic pathway genes was observed. The microarray results were validated by quantitative reverse transcription PCR with a subset of genes of interest. The XeoChips showed a chip-to-chip variation of 13.9%, compared to the 21.6% variation for spotted oligonucleotide microarrays, which is less variation than that typically reported for PCR product microarrays.

Appl Microbiol Biotechnol. 2003 May; 61(4): 342-51
Prabhu Y, Phale PS

Pseudomonas sp. strain PP2 isolated in our laboratory efficiently metabolizes phenanthrene at 0.3% concentration as the sole source of carbon and energy. The metabolic pathways for the degradation of phenanthrene, Benzoate and p-hydroxyBenzoate were elucidated by identifying metabolites, biotransformation studies, oxygen uptake by whole cells on probable metabolic intermediates, and monitoring enzyme activities in cell-free extracts. The results obtained suggest that phenanthrene degradation is initiated by double hydroxylation resulting in the formation of 3,4-dihydroxyphenanthrene. The diol was finally oxidized to 2-hydroxymuconic semialdehyde. Detection of 1-hydroxy-2-naphthoic acid, alpha-naphthol, 1,2-dihydroxy naphthalene, and salicylate in the spent medium by thin layer chromatography; the presence of 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-2,3-dioxygenase activity in the extract; O(2) uptake by cells on alpha-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate and catechol; and no O(2) uptake on o-phthalate and 3,4-dihydroxyBenzoate supports the novel route of metabolism of phenanthrene via 1-hydroxy-2-naphthoic acid --> [alpha-naphthol] --> 1,2-dihydroxy naphthalene --> salicylate --> catechol. The strain degrades Benzoate via catechol and cis,cis-muconic acid, and p-hydroxyBenzoate via 3,4-dihydroxyBenzoate and 3-carboxy- cis,cis-muconic acid. Interestingly, the culture failed to grow on naphthalene. When grown on either hydrocarbon or dextrose, the culture showed good extracellular biosurfactant production. Growth-dependent changes in the cell surface hydrophobicity, and emulsification activity experiments suggest that: (1) production of biosurfactant was constitutive and growth-associated, (2) production was higher when cells were grown on phenanthrene as compared to dextrose and Benzoate, (3) hydrocarbon-grown cells were more hydrophobic and showed higher affinity towards both aromatic and aliphatic hydrocarbons compared to dextrose-grown cells, and (4) mid-log-phase cells were significantly (2-fold) more hydrophobic than stationary phase cells. Based on these results, we hypothesize that growth-associated extracellular biosurfactant production and modulation of cell surface hydrophobicity plays an important role in hydrocarbon assimilation/uptake in Pseudomonas sp. strain PP2.

Appl Microbiol Biotechnol. 2003 Oct; 62(5-6): 579-85
Basu A, Dixit SS, Phale PS

Pseudomonas putida CSV86 metabolizes 1- and 2-methylnaphthalene through distinct catabolic and detoxification pathways. In spite of the similarity in the steps involved in the methylnaphthalene detoxification and the toluene side-chain hydroxylation pathways, the strain failed to utilize toluene or xylenes. However, it could grow on benzyl alcohol, 2- and 4-hydroxybenzyl alcohol. Metabolic studies suggest that the benzyl alcohol metabolism proceeds via the benzaldehyde, Benzoate, and catechol ortho-cleavage pathway, in contrast to the well established catechol meta-cleavage pathway. Carbon source-dependent enzyme activity studies suggest that the degradation of aromatic alcohol involves two regulons. Aromatic alcohol induces the upper regulon, which codes for aromatic alcohol- and aromatic aldehyde-dehydrogenase and converts alcohol into acid. The aromatic acid so generated induces the specific lower regulon and is metabolized via either the ortho- or the meta-cleavage pathway. CSV86 cells transform 1- and 2-methylnaphthalene to 1- and 2-hydroxymethyl naphthalene, which are further converted to the respective naphthoic acids due to the basal level expression and broad substrate specificity of the upper regulon enzymes.

FEMS Microbiol Lett. 2002 Jun 18; 212(1): 139-43
Philipp B, Kemmler D, Hellstern J, Gorny N, Caballero A, Schink B

The denitrifying bacterium Thauera aromatica strain AR-1 grows anaerobically with protocatechuate (3,4-dihydroxyBenzoate (DHB)) as sole energy and carbon source. This bacterium harbors two distinct pathways for degradation of aromatic compounds, the benzoyl-coenzyme A (CoA) pathway for Benzoate degradation and the hydroxyhydroquinone (HHQ) pathway for degradation of 3,5-DHB. In order to elucidate whether protocatechuate is degraded via the benzoyl-CoA or the HHQ pathway, induction experiments were carried out. Dense suspensions of cells grown on protocatechuate or Benzoate readily degraded Benzoate and protocatechuate but not 3,5-DHB. Dense suspensions of 3,5-DHB-grown cells degraded 3,4- and 3,5-DHB at similar rates, but Benzoate was not degraded. 3,5-DHB hydroxylating activity was found only in cells grown with this substrate. HHQ dehydrogenase activity was found in extracts of cells grown with 3,5-DHB and at a low rate also in protocatechuate-grown cells, but not in extracts of cells grown with Benzoate. Activities of protocatechuyl-CoA synthetase and protocatechuyl-CoA reductase leading to 3-hydroxybenzoyl-CoA were found in extracts of cells grown with protocatechuate. There was no repression of the HHQ pathway by the presence of protocatechuate, unlike by degradation of Benzoate. We conclude that protocatechuate is not degraded via the HHQ pathway because there was no evidence of a hydroxylation reaction involved in this process. Instead, our results strongly suggest that protocatechuate is degraded via a pathway which connects to the benzoyl-CoA route of degradation.

J Bacteriol. 2001 Mar; 183(6): 1899-908
Mohamed ME, Zaar A, Ebenau-Jehle C, Fuchs G

The aerobic metabolism of Benzoate in the proteobacterium Azoarcus evansii was reinvestigated. The known pathways leading to catechol or protocatechuate do not operate in this bacterium. The presumed degradation via 3-hydroxybenzoyl-coenzyme A (CoA) and gentisate could not be confirmed. The first committed step is the activation of Benzoate to benzoyl-CoA by a specifically induced Benzoate-CoA ligase (AMP forming). This enzyme was purified and shown to differ from an isoenzyme catalyzing the same reaction under anaerobic conditions. The second step postulated involves the hydroxylation of benzoyl-CoA to a so far unknown product by a novel benzoyl-CoA oxygenase, presumably a multicomponent enzyme system. An iron-sulfur flavoprotein, which may be a component of this system, was purified and characterized. The homodimeric enzyme had a native molecular mass of 98 kDa as determined by gel filtration and contained 0.72 mol flavin adenine dinucleotide (FAD), 10.4 to 18.4 mol of Fe, and 13.3 to 17.9 mol of acid-labile sulfur per mol of native protein, depending on the method of protein determination. This Benzoate-induced enzyme catalyzed a benzoyl-CoA-, FAD-, and O2-dependent NADPH oxidation surprisingly without hydroxylation of the aromatic ring; however, H2O2 was formed. The gene (boxA, for Benzoate oxidation) coding for this protein was cloned and sequenced. It coded for a protein of 46 kDa with two amino acid consensus sequences for two [4Fe-4S] centers at the N terminus. The deduced amino acid sequence showed homology with subunits of ferredoxin-NADP reductase, nitric oxide synthase, NADPH-cytochrome P450 reductase, and phenol hydroxylase. Upstream of the boxA gene, another gene, boxB, encoding a protein of 55 kDa was found. The boxB gene exhibited homology to open reading frames in various other bacteria which code for components of a putative aerobic phenylacetyl-CoA oxidizing system. The boxB gene product was one of at least five proteins induced when A. evansii was grown on Benzoate.

Biol Chem. 1998 Aug-Sep; 379(8-9): 1201-5
Arteel GE, Mostert V, Oubrahim H, Briviba K, Abel J, Sies H

In order to study functions of selenoprotein P in human plasma, its level was lowered via two techniques, chromatography on Sepharose-bound heparin, or immunoprecipitation; Western blot analysis showed that both techniques were effective at substantially lowering selenoprotein P levels in plasma. When peroxynitrite was infused to maintain a 0.9 microM steady-state concentration, plasma made deficient in selenoprotein P diminished Benzoate hydroxylation significantly less than control plasma. Similar differences were found for protein 3-nitrotyrosine formation, determined by Western blot analysis. Conversely, in a selenoprotein P-enriched plasma preparation obtained via heparin-Sepharose chromatography, protection against Benzoate hydroxylation was above controls. Likewise, a supernatant from control plasma that had been exposed to anti-selenoprotein P antibodies was less efficient in preventing oxidation and nitration reactions of peroxynitrite than the supernatant from plasma exposed to a non-specific antibody (rabbit anti-sheep IgG). These data demonstrate a role of selenoprotein P in human plasma in the defense against peroxynitrite.

Eur J Biochem. 1995 Jan 15; 227(1-2): 161-8
Niemetz R, Altenschmidt U, Brucker S, Fuchs G

A new variant of aerobic Benzoate degradation has been found in a denitrifying bacterium in which benzoyl-CoA is the first intermediate [Altenschmidt, U., Oswald, B., Steiner, E., Herrmann, H. & Fuchs, G. (1993) New aerobic Benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp, J. Bacteriol. 175, 4851-4858)]. The initial reaction is catalyzed by Benzoate-CoA ligase (AMP-forming), converting Benzoate into benzoyl-CoA. The next step is 3-hydroxylation of benzoyl-CoA to 3-hydroxybenzoyl-CoA catalyzed by a flavin-nucleotide-dependent monooxygenase, benzoyl-CoA 3-monooxygenase. This novel enzyme has been purified and studied. It is specific for NADPH and requires the presence of a flavin nucleotide for activity; both FAD or FMN function similarly well as cofactor. Only benzoyl-CoA, but not Benzoate, is hydroxylated. The protein is a monomer of M(r) 65,000 and is induced when cells are grown aerobically with Benzoate. 3-Hydroxybenzoyl-CoA is further hydroxylated para to the hydroxyl group affording 2,5-dihydroxyBenzoate (gentisate). This reaction requires another monooxygenase, 3-hydroxybenzoyl-CoA 6-monooxygenase, which is unspecific specific with respect to the pyridine nucleotide. Cells contain a second 6-monooxygenase activity which acts on free 3-hydroxyBenzoate. Based on these and other data, the outlines of the new aerobic Benzoate pathway have been deduced. The proposed intermediates are benzoyl-CoA, 3-hydroxybenzoyl-CoA, gentisate, maleylpyruvate, fumarylpyruvate and fumarate plus pyruvate.

J Biol Chem. 1994 Oct 21; 269(42): 26066-75
Rubbo H, Radi R, Trujillo M, Telleri R, Kalyanaraman B, Barnes S, Kirk M, Freeman BA

Superoxide (O2-.), nitric oxide (.NO), and their reaction product peroxynitrite (ONOO-) have all been shown to independently exert toxic target molecule reactions. Because these reactive species are often generated in excess during diverse inflammatory and other pathologic circumstances, we assessed the influence of .NO on membrane lipid peroxidation induced by O2-., H2O2, and .OH derived from xanthine oxidase (XO) and by ONOO-. Experimental conditions in lipid oxidation systems were adjusted to yield different rates of delivery of .NO, relative to rates of O2-. and H2O2 generation, by infusion of either .NO or via .NO released from S-nitroso-N-acetylpenicillamine or S-nitrosoglutathione. Peroxidation of phosphatidylcholine liposomes was assessed by formation of thiobarbituric acid-reactive products and by liquid chromatography-mass spectrometry. Liposomes exposed to XO-derived reactive species in the presence of .NO exhibited both stimulation and inhibition of lipid peroxidation, depending on the ratio of the rates of reactive oxygen species production and .NO introduction into reaction systems. Nitric oxide alone did not induce lipid peroxidation. Linolenic acid emulsions peroxidized by XO-derived reactive species showed similar dose-dependent regulation of lipid peroxidation by .NO. Mass spectral analysis of oxidation products showed formation of nitrito-, nitro-, nitrosoperoxo-, and/or nitrated lipid oxidation adducts, demonstrating that .NO serves as a potent terminator of radical chain propagation reactions. Electron spin resonance (ESR) analysis of incubation mixtures provided no evidence for formation of paramagnetic iron-lipid-nitric oxide complexes in reaction systems. Peroxynitrite-dependent lipid peroxidation, which predominantly occurs by metal-independent mechanisms, was also inhibited by .NO. Peroxynitrite-mediated Benzoate hydroxylation was partially inhibited by .NO, inferring reaction between .NO and ONOOH. It is concluded that .NO can both stimulate O2-./H2O2/.OH-induced lipid oxidation and mediate oxidant-protective reactions in membranes at higher rates of .NO production, with the prooxidant versus antioxidant outcome critically dependent on relative concentrations of individual reactive species. Prooxidant reactions of .NO will occur after O2-. reaction with .NO to yield potent secondary oxidants such as ONOO- and the antioxidant effects of .NO a consequence of direct reaction with alkoxyl and peroxyl radical intermediates during lipid peroxidation, thus terminating lipid radical chain propagation reactions.

Arch Microbiol. 1991; 156(2): 152-8
Altenschmidt U, Fuchs G

The anaerobic degradation of toluene has been studied with whole cells and by measuring enzyme activities. Cultures of Pseudomonas strain K 172 were grown in mineral medium up to a cell density of 0.5 g of dry cells per liter in fed-batch culture with toluene and nitrate as the sole carbon and energy sources. A molar growth yield of 57 g of cell dry matter formed per mol toluene totally consumed was determined. The mean generation time was 24 h. The redox balance between toluene consumed (oxidation and cell material synthesis) and nitrate consumed (reduction to nitrogen gas and assimilation as NH3) was 77% of expectation if toluene was completely oxidized; this indicated that the major amount of toluene was mineralized to CO2. It was tested whether the initial reaction in anaerobic toluene degradation was a carboxylation or a dehydrogenation (anaerobic hydroxylation); the hypothetical carboxylated or hydroxylated intermediates were tested with whole cells applying the method of simultaneous adaptation; cells pregrown on toluene degraded benzyl alcohol, benzaldehyde, and benzoic acid without lag, 4-hydroxyBenzoate and p-cresol with a 90 min lag phase, and phenylacetate after a 200 min lag phase. The cells were not at all adapted to degrade 2-methylBenzoate, 4-methylBenzoate, o-cresol, and m-cresol, nor did these compounds support growth within a few days after inoculation with cells grown on toluene. In extracts of cells anaerobically grown on toluene, benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase, and benzoyl-CoA synthetase (AMP forming) activities were present. The data (1) conclusively show anaerobic growth of a pure culture on toluene; (2) suggest that toluene is anaerobically degraded via benzoyl-CoA; (3) imply that water functions as the source of the hydroxyl group in a toluene methylhydroxylase reaction.

Appl Environ Microbiol. 1984 May; 47(5): 947-51
Massé R, Messier F, Péloquin L, Ayotte C, Sylvestre M

The biodegradation products of 4-chlorobiphenyl were analyzed in an Achromobacter sp. strain and a Bacillus brevis strain. Both strains generated the same metabolites, with 4-chlorobenzoic acid as the major metabolic product. Our results corroborate previous observations whereby most bacterial strains degrade the chlorobiphenyls via a major pathway which proceeds by an hydroxylation in position 2,3 and a meta-1,2 fission. However, we also detected several metabolites whose structure suggests the existence of other routes for the degradation of chlorinated biphenyls.

J Bacteriol. 1984 Apr; 158(1): 79-83
Hughes EJ, Bayly RC, Skurray RA

A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways. Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway. The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates. Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions. Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes. hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme.

Xenobiotica. 1980 May; 10(5): 355-64
Crayford JV, Hutson DH

1. 3-Phenoxy[14C]benzoic acid administered orally to rats (0.76 to > 100 mg/kg) is extensively metabolized and rapidly eliminated mostly via the urine. 2. The major metabolic pathway involves 4'-hydroxylation followed by conjugation of the resulting phenol with sulphate. Only minor amounts of amino acid and glucuronic acid conjugation were observed. 3. 3-Phenoxy[14C]benzoyl glucoside, derived from the metabolism of the acid by corn leaves, was also rapidly absorbed by rats and eliminated as a mixture of metabolites very similar to that derived from 3-phenoxybenzoic acid. It was concluded that the glucoside conjugate was rapidly hydrolysed to the free acid in vivo.

Appl Microbiol. 1974 Feb; 27(2): 360-7
Kirk TK, Lorenz LF

The degradation of several alkyl ethers of vanillic acid, of 3-ethoxy-4-hydroxybenzoic acid, and of syringic acid, by the lignin-decomposing fungus Polyporus dichrous included (i) 4-dealkylation (e.g., 3-ethoxy-4-isopropoxybenzoic acid was in part dealkylated to 3-ethoxy-4-hydroxybenzoic acid), (ii) hydroxylation of the 4-alkoxyl groups (e.g., 3-ethoxy-4-isopropoxybenzoic acid was oxidized in part to 2-[4-carboxy-2-ethoxyphenoxy]-propane-1-ol), and (iii) reduction of carboxyl groups (older cultures) (e.g., 3-ethoxy-4-isopropoxybenzoic acid was reduced to 3-ethoxy-4-isopropoxybenzaldehyde and 3-ethoxy-4-isopropoxybenzyl alcohol). Some ethers (e.g., tri-O-methyl gallic acid and glycerol-beta-[4-carboxy-2-ethoxyphenyl]-ether) were not affected. The dealkylations and hydroxylations indicate that the fungus has a relatively nonspecific mechanism for oxygenating various 4-alkoxyl groups of alkoxybenzoic acids; no evidence for oxygenation of 3-alkoxyl groups was obtained. hydroxylation products were generally degraded further, probably via dealkylation. The vanillic acid and 3-ethoxy-4-hydroxybenzoic acid formed by dealkylations were readily metabolized. Although the isopropyl ether of syringic acid was hydroxylated to 2-(4-carboxy-2, 6-dimethoxyphenoxy)-propane-1-ol, neither this compound nor the parent isopropyl ether was dealkylated; syringic acid itself was only slowly and incompletely metabolized. The relationship of these results to lignin degradation is discussed.

Biochem J. 1968 Jan; 106(1): 1-13
Nery R

1. Urethane and N-hydroxyurethane are interconvertible in C(-) and C57 mice. 2. In newborn C57/DBA hybrid mice, prior treatment with 3-methylcholanthrene or urethane stimulated the N-hydroxylation of urethane; SKF 525A inhibited the N-hydroxylation at 24hr. but stimulated it at 48hr. after administration. 3. Liver homogenates of CBA and C3H mice, and of Chester Beatty and hooded rats, but not whole-body homogenates of 1-day-old C57/DBA mice or lung homogenate of 3-week-old Chester Beatty rats, metabolized urethane into N-hydroxyurethane in small but definite amounts. 4. Nitrite was detected in the bodies of newborn C57/DBA hybrid mice treated with lethal doses of urethane or N-hydroxyurethane; nitrite formation from N-hydroxyurethane was stimulated by pretreatment of the animals with 3-methylcholanthrene. 5. The rate of catabolism of N-hydroxyurethane by C57/DBA mice was faster in 8-day-old than in 1-day-old animals of the same sex, and faster in females than in males of the same age. 6. Liver slices of several species of rats and mice catabolized N-hydroxyurethane at rates that varied with the age and sex of animals of the same species; liver homogenates or microsomes were less effective than slices from the same liver. 7. The enzyme activity was destroyed by boiling or freezing the liver; it was inhibited by increasing substrate concentration and by urethane, n-butyl carbamate, cyanide, p-benzoquinone or 2,4-dinitrophenol, but not by p-chloromercuriBenzoate or menadione. 8. The catabolism of N-hydroxyurethane by liver slices from adult H-strain rats was not oxygen-dependent. 9. Lung homogenates of 4-week-old female Chester Beatty rats catabolized N-hydroxyurethane at 40% of the rate of liver slices from the same source. 10. O-Acetyl- and O-ethoxycarbonyl-N-hydroxyurethane were rapidly deacylated by liver homogenates from adult hooded rats and adult C57 mice, and by human erythrocytes. 11. N-Hydroxyurethane reacted rapidly with pyridoxal phosphate at pH7.4 and 37 degrees . 12. The rate of decomposition of N-hydroxyurethane in 0.1 n-sodium hydroxide was increased by Ni(2+), Cu(2+), Mn(2+) and [Fe(CN)(6)](3-) and decreased by Cr(2+), Zn(2+), Co(2+), Mg(2+) and Fe(2+). 13. Attempts to synthesize sulphonates of N-hydroxyurethane gave ethyl hydrogen sulphate, probably via rearrangement of the unstable O-sulphonate.

Biochem J. 1965 Apr; 95: 70-6
ELLIOTT TH, TAO RC, WILLIAMS RT

1. When [U-(14)C]methylcyclohexane is fed to rabbits (dose 2-2.5m-moles/kg. body wt.), 65% of the radioactivity is excreted in the urine as metabolites, 0.5% appears in the faeces and about 15% in the expired air, some 4-5% remaining in the body in about 60hr. after dosing. The 15% of the dose appearing in the expired air consists of unchanged methylcyclohexane (10%) and (14)CO(2) (5%). The low output of (14)CO(2) shows that reactions leading to complete oxidation of methylcyclohexane are of minor importance. 2. The main metabolite found in the urine was the glucuronide of trans-4-methylcyclohexanol which was isolated. Seven methylcyclohexanols were found in the urine as conjugated glucuronides. The amounts of these were determined by isotope dilution to be as follows: cis-2-, 0.6%; trans-2-, 1.2%; cis-3-, 11.5%; trans-3-, 10.5%; cis-4-, 2.4%; trans-4-methylcyclohexanol, 14.7%, cyclohexylmethanol, 0.3%. No 1-methylcyclohexanol was found. There was evidence also that a small amount (approx. 1%) of the hydrocarbon aromatized to benzoic acid, probably via cyclohexylmethanol and cyclohexane-carboxylic acid. 3. The pattern of hydroxylation and the various amounts of the isomers found suggest that the hydroxylation in vivo of methylcyclohexane is dependent on steric factors in the molecule, hydroxylation occurring to the greatest extent at the carbon atom furthest away from the methyl group.