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PubMed2006

Structural and biochemical characterization of gentisate 1,2-dioxygenase from Escherichia coli O157:H7.

Gentisic acid (2,5-dihydroxybenzoic acid) is a key intermediate in aerobic bacterial pathways that are responsible for the metabolism of a large number of aromatic compounds. The critical step of these pathways is the oxygen-dependent reaction catalysed by gentisate 1,2-dioxygenase which opens the aromatic ring of gentisate to form maleylpyruvate. From gentisic acid, the cell derives carbon and energy through the conversion of maleylpyruvate to central metabolites. We have confirmed the annotation of a gentisate 1,2-dioygenase from the pathogenic O157:H7 Escherichia coli strain and present the first structural characterization of this family of enzymes. The identity of the reaction product was revealed using tandem mass spectroscopy. The operon responsible for the degradation of gentisate in this organism exhibits a high degree of conservation with the gentisate-degrading operons of other pathogenic bacteria, including the Shiga toxin-producing E. coli O103:H2, but does not appear to be present in non-pathogenic strains. The acquisition of the gentisate operon may represent a special adaptation to meet carbon source requirements under conditions of environmental stress and may provide a selective advantage for enterohaemorrhagic E. coli relative to their non-pathogenic counterparts.

Amino Acid Sequence↗
PubMed2007

Proteome analysis of heat shock protein expression in Pseudomonas alcaligenes NCIMB 9867 in response to gentisate exposure and elevated growth temperature.

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) degrades xylenols and cresols via the gentisate pathway. P25X expresses two isofunctional gentisate 1,2-dioxygenases (GDO I and GDO II). The expression of both GDOs was not detected when P25X cells were grown at 42 degrees C, even in the presence of gentisate. A total of 19 heat shock proteins (Hsps) belonging to the Hsp100, Hsp90, Hsp70, Hsp60, Hsp45, and small heat shock protein (sHsp) families were identified among the protein spots that were either newly detected or were expressed at levels of at least twofold higher when P25X cells were cultured at 32 or 42 degrees C in the presence and absence of gentisate. Among these, 16 Hsps were commonly expressed at 42 degrees C. Two additional Hsps (H5 and H13) from the Hsp90 and Hsp60 families, respectively, were expressed only when P25X cells were grown at 42 degrees C and in the presence of gentisate. A protein of the sHsp (H16) family was expressed only in the presence of gentisate at 32 degrees C but not at 42 degrees C. The GroEL chaperonins of the Hsp60 family comprised the largest group of Hsps identified and exhibited high level of expression at 42 degrees C following gentisate exposure.

Gene Expression Regulation, Bacterial↗
PubMed2006

Heterologous expression and localization of gentisate transporter Ncg12922 from Corynebacterium glutamicum ATCC 13032.

Ralstonia sp. strain U2 metabolizes naphthalene via gentisate (2,5-dihydroxybenzoate) to central metabolites, but it was found unable to utilize gentisate as growth substrate. A putative gentisate transporter encoded by ncg12922 from Corynebacterium glutamicum ATCC 13032 was functionally expressed in Ralstonia sp. strain U2, converting strain U2 to a gentisate utilizer. After ncg12922 was inserted into plasmid pGFPe with green fluorescence protein gene gfp, the expressed fusion protein Ncg12922-GFP could be visualized in the periphery of Escherichia coli cells under confocal microscope, consistent with a cytoplasmic membrane location. In contrast, GFP was ubiquitous in the cytoplasm of E. coli cells carrying pGFPe only. Gentisate 1,2-dioxygenase activity was present in the cell extract from strain U2 induced with gentisate but at a much lower level (one-fifth) than that obtained with salicylate. However, it exhibited a similar level in strain U2 containing Ncg12922 induced either by salicylate or gentisate.

Bacterial Proteins↗
PubMed2005

Gentisic acid, an aspirin metabolite, inhibits oxidation of low-density lipoprotein and the formation of cholesterol ester hydroperoxides in human plasma.

Gentisic acid, an aspirin metabolite, has an antioxidant effect, although its detailed mechanism remains elusive. The present study was designed to determine whether it inhibits low-density lipoprotein (LDL) oxidation and the formation of lipid hydroperoxides in human plasma. The susceptibility of LDL oxidative modification was investigated by a method using 2,2'-azobis or Cu2+. To study the effect of gentisic acid on free radical-induced damage to plasma lipids, cholesterol ester hydroperoxides generated by incubating human fresh plasma with Cu2+ and gentisic acid was analyzed. Gentisic acid inhibited LDL oxidation in a concentration-dependent manner. It significantly inhibited the formation of cholesterol ester hydroperoxides in plasma, and was consumed after the depletion of ascorbic acid and reduced form of coenzyme Q-10 (CoQH2-10), whereas concentrations of other antioxidants remained unchanged. Gentisic acid had a potent free radical scavenging activity with a minimal chelating effect. The potent antioxidant property of gentisic acid may partly account for the anti-atherogenic effects of aspirin.

Antioxidants↗
PubMed2001

nag genes of Ralstonia (formerly Pseudomonas) sp. strain U2 encoding enzymes for gentisate catabolism.

Ralstonia sp. strain U2 metabolizes naphthalene via gentisate to central metabolites. We have cloned and sequenced a 21.6-kb region spanning the nag genes. Upstream of the pathway genes are nagY, homologous to chemotaxis proteins, and nagR, a regulatory gene of the LysR family. Divergently transcribed from nagR are the genes for conversion of naphthalene to gentisate (nagAaGHAbAcAdBFCQED) (S. L. Fuenmayor, M. Wild, A. L. Boyes, and P. A. Williams, J. Bacteriol. 180:2522-2530, 1998), which except for the insertion of nagGH, encoding the salicylate 5-hydroxylase, are homologous to and in the same order as the genes in the classical upper pathway operon described for conversion of naphthalene to salicylate found in the NAH7 plasmid of Pseudomonas putida PpG7. Downstream of nahD is a cluster of genes (nagJIKLMN) which are probably cotranscribed with nagAaGHAbAcAdBFCQED as a single large operon. By cloning into expression vectors and by biochemical assays, three of these genes (nagIKL) have been shown to encode the enzymes involved in the further catabolism of gentisate to fumarate and pyruvate. NagI is a gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate and is also able to catalyze the oxidation of some substituted gentisates. NagL is a reduced glutathione-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate. NagK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The three other genes (nagJMN) have also been cloned and overexpressed, but no biochemical activities have been attributed to them. NagJ is homologous to a glutathione S-transferase, and NagM and NagN are proteins homologous to each other and to other proteins of unknown function. Downstream of the operon is a partial sequence with homology to a transposase.

Bacterial Proteins↗
PubMed1985

Inhibition of cyclooxygenase mediated by electrochemical oxidation of gentisic acid.

Prostaglandin synthase (EC 1.14.99.1, 8,11,14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase), commonly referred to as cyclooxygenase, was inhibited irreversibly upon application of a fixed oxidative potential (+0.4 V versus saturated calomel electrode) in the presence of the aspirin metabolite gentisic acid (2,5-dihydroxybenzoic acid). Electrolyses were carried out at 0 degrees C in a phosphate buffer solution (pH 7.2, 0.1 M) using a carbon felt electrode. This electroinactivation process was time-dependent and pseudo first-order with respect to gentisic acid at concentrations up to 300 microM. These concentrations of gentisic acid are below those normally reported to be inhibitory. The enzyme was stable to this applied potential in the absence of gentisic acid. Similar treatment of apoenzyme (heme-removed) revealed no loss in catalytic activity after reconstitution to the holoenzyme. Oxyphenbutazone, a nonoxidizable competitive inhibitor of cyclolooxygenase, was observed to protect the enzyme from electrolytic inactivation mediated by gentisic acid. Radiolabeling studies indicated the covalent attachment of approximately 1 eq of gentisic acid/subunit of enzyme. These studies support the possible role of quinonoid intermediates in the observed anti-inflammatory action of salicylate derivatives.

Animals↗
PubMed2005

Proteome investigation of the global regulatory role of sigma 54 in response to gentisate induction in Pseudomonas alcaligenes NCIMB 9867.

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) utilizes the gentisate pathway for the degradation of aromatic hydrocarbons. The gene encoding the alternative sigma (sigma) factor sigma(54), rpoN, was cloned from strain P25X and a rpoN knock-out strain, designated G54, was constructed by insertional inactivation with a kanamycin resistance gene cassette. The role of sigma(54) in the physiological response of P. alcaligenes P25X to gentisate induction was assessed by comparing the global protein expression profiles of the wild-type P25X with the rpoN mutant strain G54. Analysis of two-dimensional polyacrylamide gel electrophoresis gels showed that 39 out of 355 prominent protein spots exhibited differential expression as a result of the insertional inactivation of rpoN. Identification of the protein spots by matrix-assisted laser desorption/ionization-time of flight/time of flight revealed a wide diversity of proteins that are affected by the sigma(54) mutation, the largest group being proteins that are involved in carbon metabolism. The strictly inducible gentisate 1,2-dioxygenase, one of two isofunctional copies of the key enzyme in the gentisate pathway, and enzymes of the TCA cycle, pyruvate metabolism and gluconeogenesis were part of this group. Other proteins that are part of the sigma(54) regulon include enzymes implicated in nitrogen metabolism, transport proteins, stress-response proteins and proteins involved in cell motility. The results of this study showed that sigma(54) plays a global regulatory role in the expression of a wide variety of genes in P. alcaligenes, including the wild-type response to the presence of the aromatic inducer, gentisate.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

The gene ncgl2918 encodes a novel maleylpyruvate isomerase that needs mycothiol as cofactor and links mycothiol biosynthesis and gentisate assimilation in Corynebacterium glutamicum.

Data mining of the Corynebacterium glutamicum genome identified 4 genes analogous to the mshA, mshB, mshC, and mshD genes that are involved in biosynthesis of mycothiol in Mycobacterium tuberculosis and Mycobacterium smegmatis. Individual deletion of these genes was carried out in this study. Mutants mshC- and mshD- lost the ability to produce mycothiol, but mutant mshB- produced mycothiol as the wild type did. The phenotypes of mutants mshC- and mshD- were the same as the wild type when grown in LB or BHIS media, but mutants mshC- and mshD- were not able to grow in mineral medium with gentisate or 3-hydroxybenzoate as carbon sources. C. glutamicum assimilated gentisate and 3-hydroxybenzoate via a glutathione-independent gentisate pathway. In this study it was found that the maleylpyruvate isomerase, which catalyzes the conversion of maleylpyruvate into fumarylpyruvate in the glutathione-independent gentisate pathway, needed mycothiol as a cofactor. This mycothiol-dependent maleylpyruvate isomerase gene (ncgl2918) was cloned, actively expressed, and purified from Escherichia coli. The purified mycothiol-dependent isomerase is a monomer of 34 kDa. The apparent Km and Vmax values for maleylpyruvate were determined to be 148.4 +/- 11.9 microM and 1520 +/- 57.4 micromol/min/mg, respectively (mycothiol concentration, 2.5 microM). Previous studies had shown that mycothiol played roles in detoxification of oxidative chemicals and antibiotics in streptomycetes and mycobacteria. To our knowledge, this is the first demonstration that mycothiol is essential for growth of C. glutamicum with gentisate or 3-hydroxybenzoate as carbon sources and the first characterization of a mycothiol-dependent maleylpyruvate isomerase.

Amino Acid Sequence↗
PubMed1988

Regulation of isofunctional enzymes in Pseudomonas alcaligenes mutants defective in the gentisate pathway.

The regulation of the inducible set of gentisate pathway enzymes used by Pseudomonas alcaligenes (P25X1) has been studied in strains derived from mutant strains of P25X1 that had lost the constitutive enzymes that degrade m-cresol, 2,5-xylenol and 3,5-xylenol. The enzyme, 3-hydroxybenzoate 6-hydroxylase II, that catalyzes the oxidation of 3-hydroxybenzoate to gentisate is substrate- and product-induced while gentisate dioxygenase II is substrate induced. Neither 3-hydroxybenzoate nor gentisate could induce the synthesis of maleylpyruvate hydrolase II and fumarylpyruvate hydrolase II. The results suggest that the structural genes encoding these four inducible enzymes and malepylpyruvate hydrolase I (a constitutive enzyme) exist in at least four operons. There is strict induction specificity of expression of this inducible set of gentisate pathway enzymes. 3-Hydroxy-4-methylbenzoate failed to induce whilst 3-hydroxybenzoate and 3-hydroxy-5-methylbenzoate served as inducers of 6-hydroxylase II. Degradation of 2,5-xylenol is mediated by constitutive enzymes whereas the inducible set of enzymes are responsible for the metabolism of m-cresol and 3,5-xylenol.

Enzyme Induction↗
PubMed1975

Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis.

Gentisate:oxygen 1,2-oxidoreductase (decyclizing) (EC 1.13.11.4; gentisate 1,2-dioxygenase) from Moraxella osloensis was purified to homogeneity as shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 154,000 and gives rise to subunits of molecular weight 40,000 in the presence of sodium dodecyl sulfate. Gentisate 1,2-dioxygenase showed broad substrate specificity and attacked a range of halogen- and alkyl-substituted gentisic acids. Maleylpyruvate, the product formed from gentisate, was degraded by cell extracts supplemented with reduced glutathione, but substituted maleylpyruvates were not attacked under these conditions.

Ammonium Sulfate↗
PubMed1980

Evidence for isofunctional enzymes used in m-cresol and 2,5-xylenol degradation via the gentisate pathway in Pseudomonas alcaligenes.

Study of the reaction sequence by which Pseudomonas alcaligenes (P25X1) and derived mutants degrade m-cresol, 2,5-xylenol, and their catabolites has provided indirect evidence for the existence of two or more isofunctional enzymes at three different steps. Maleylpyruvate hydrolase activity appears to reside in two different proteins with different specificity ranges, one of which (MPH1) is expressed constitutively; the other (MPH11) is strictly inducible. Two gentisate 1,2-dioxygenase activities were found, one of which is constitutively expressed and possesses a broader specificity range than the other, which is inducible. From oxidation studies with intact cells, there appear to be two activities responsible for the 6-hydroxylation of 3-hydroxybenzoate, and again a broadly specific activity is present regardless of growth conditions; the other is inducible by 3-hydroxybenzoate. Three other enzyme activities are also detected in uninduced cells, viz., xylenol methylhydroxylase, benzylalcohol dehydrogenase, and benzaldehyde dehydrogenase. All apparently possess broad specificity. Fumarylpyruvate hydrolase was also detected but only in cells grown with m-cresol, 3-hydroxybenzoate, or gentisate. Mutants, derived either spontaneously or after treatment with mitomycin C, are described, certain of which have lost the ability to grow with m-cresol and 2,5-xylenol and some of which have also lost the ability to form the constitutive xylenol methylhydroxylase, benzylalcohol dehydrogenase, benzaldehyde dehydrogenase, 3-hydroxybenzoate 6-hydroxylase, and gentisate 1,2-dioxygenase. Such mutants, however, retain ability to synthesize inducibly a second 3-hydroxybenzoate 6-hydroxylase and gentisate 1,2-dioxygenase, as well as maleylpyruvate hydrolase (MPH11) and fumarylpyruvate hydrolase; MPH1 was still synthesized. These findings suggest the presence of a plasmid for 2,5-xylenol degradation which codes for synthesis of early degradative enzymes. Other enzymes, such as the second 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, maleylpyruvate hydrolase (MPH1 and MPH11), and fumarylpyruvate hydrolase, appear to be chromosomally encoded and, with the exception of MPH1, strictly inducible.

Alcohol Oxidoreductases↗
PubMed1998

A gene cluster encoding steps in conversion of naphthalene to gentisate in Pseudomonas sp. strain U2.

Pseudomonas sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. The genes for naphthalene dioxygenase were cloned from the plasmid DNA of strain U2 on an 8.3-kb BamHI fragment. The genes for the naphthalene dioxygenase genes nagAa (for ferredoxin reductase), nagAb (for ferredoxin), and nagAc and nagAd (for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations and by nucleotide sequencing. The genes for nagB (for naphthalene cis-dihydrodiol dehydrogenase) and nagF (for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between nagAa and nagAb were two open reading frames, homologs of which have also been identified in similar locations in two nitrotoluene-using strains (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 181:57-61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926-4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet. Eng. 19:257-269, 1997). Recombinant Escherichia coli strains with plasmids carrying this region were able to convert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance. The first open reading frame, designated nagG, encodes a protein with characteristics of a Rieske-type iron-sulfur center homologous to the large subunits of dihydroxylating dioxygenases, and the second open reading frame, designated nagH, encodes a protein with limited homology to the small subunits of the same dioxygenases. Cloned together in E. coli, nagG, nagH, and nagAb, were able to convert salicylate (2-hydroxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore encode a salicylate 5-hydroxylase activity. Single-gene knockouts of nagG, nagH, and nagAb demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunits of salicylate 5-hydroxylase linked to an electron transport chain consisting of NagAb and NagAa, although E. coli appears to be able to partially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization. However growth on both naphthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2. Sequence comparisons suggest that the novel gene order nagAa-nagG-nagH-nagAb-nagAc-nagAd-++ +nagB-nagF represents the archetype for naphthalene strains which use the gentisate pathway rather than the meta cleavage pathway of catechol.

Base Sequence↗
PubMed1980

Gentisic acid: a new stabilizer for low tin skeletal imaging agents: concise communication.

In vitro stabilization of low-tin bone-imaging agents has previously been achieved with ascorbic acid. In this study gentisic acid is shown to be an equally effective antioxidant for the (1-hydroxyethylidene) diphosphonate (HEDP) and hydroxymethylenediphosphonate (HMDP) skeletal agents. In vitro studies show less than 2% free sodium [99mTc] pertechnetate at 24 hr with the gentisic acid stabilizer. Studies in guinea pigs at 3 and 24 hr--whether with C-14 or H-3-labeled gentisic acid as stabilizer--show no alteration in the biodistribution of either skeletal imaging agent by the addition of the gentisic acid. Gentisic acid is a safe and effective stabilizer, and clinical studies have shown bioequivalency with ascorbic acid.

Animals↗
PubMed1990

In vitro comparison of HMPAO and gentisic acid for labelling leukocytes with 99mTc.

Leukocytes can be labelled with 99mTc using HMPAO and gentisic acid methods. We compared the two methods with respect to labelling efficiency on mixed leukocytes and isolated polymorphonuclear (PMN) and mononuclear (MN) cells, and the in vitro stability of the label. HMPAO produced approximately 70% labelling efficiency on mixed or PMN cells and the label was stable in saline or plasma. Labelling efficiency on MN was only 14% and was less stable. Gentisic acid produced a labelling efficiency of 52% on PMN and 35% on MN; both were stable in saline but less stable in plasma. In conclusion, HMPAO produces higher labelling efficiency and the label shows greater in vitro stability in plasma. However, gentisic acid is much less expensive to use, allows labelling of MN cells, and should result in more favourable microdosimetry. Preliminary clinical results suggest that gentisic acid is equivalent to HMPAO but has the advantage of being much cheaper.

Costs and Cost Analysis↗
PubMed2003

Molecular characterization of an inducible gentisate 1,2-dioxygenase gene, xlnE, from Pseudomonas alcaligenes NCIMB 9867.

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) produces isofunctional enzymes of the gentisate pathway that enables the degradation of xylenols and cresols via gentisate. Previous reports had indicated that one set of enzymes is constitutively expressed whereas the other set is strictly inducible by aromatic hydrocarbon substrates. The gene encoding gentisate 1,2-dioxygenase (GDO), the enzyme that catalyzes the cleavage of the gentisate aromatic ring, was cloned from strain P25X. The GDO gene, designated xlnE, is 1,044 bp, and is part of a 5.4 kb operon which consists of six genes, xlnEFGHID. Transcription of this operon was driven by a sigma 70-type promoter, PxlnE, located 123 bp upstream of the xlnE start codon. Primer extension analysis showed that the xlnE transcription start point is located at the -87 adenine residue. In a P25X xlnE knockout mutant, GDO activity could only be detected when cells were grown in the presence of aromatic substrates, suggesting that xlnE encodes for the constitutive copy of GDO. This was verified by constructing a P25X strain with xlnE transcriptionally fused to a promoterless catechol 2,3-dioxygenase gene. In this strain, catechol 2,3-dioxygenase activity was detected in cells that were grown in the absence of aromatic inducers. However, catechol 2,3-dioxygenase activity increased up to four fold when these cells were grown in the presence of aromatic substrates, in particular 3-hydroxybenzoate. Thus, xlnE is in fact, inducible and the constitutive activity observed under non-inducing conditions was due to its relatively high basal levels of expression.

Amino Acid Sequence↗
PubMed2001

Determination of salicylate, gentisic acid and salicyluric acid in human urine by capillary electrophoresis with laser-induced fluorescence detection.

Acetylsalicylic acid (Aspirin) is rapidly metabolized to salicylic acid (salicylate) and other compounds, including gentisic acid and salicyluric acid. Monitoring of salicylate and its metabolites is of toxicological, pharmacological and biomedical interest. Three capillary electrophoresis (CE) methods featuring alkaline aqueous buffers, laser-induced fluorescence (LIF) detection and no solute extraction or derivatization have been explored. A competitive binding, electrokinetic capillary-based immunoassay is developed that recognizes the presence of salicylate and gentisic acid in urine. Differentiation of the two compounds, however, is problematic. With appropriate ultraviolet excitation, many salicylate-related compounds are fluorescent so that CE with direct urine injection and LIF detection permits the determination of salicylate, gentisic acid and salicyluric acid. Using a HeCd laser with 325 nm produces interference-free monitoring of all three compounds. Using 257 nm excitation from a frequency doubled Ar ion laser, native fluorescence of an endogenous urinary compound that co-migrates with gentisic acid is observed. With wavelength-resolved fluorescence detection, however, the two substances are distinguished. Furthermore, this technique, with comparison to literature data, permits the putative assignment of several peaks to other salicylate metabolites, namely glucuronide conjugates of salicylate and salicyluric acid. All three CE-LIF techniques have been applied to toxicological patient urines and urines collected after ingestion of 500 mg acetylsalicylic acid. CE results compare favorably with those obtained by a commercial fluorescence polarization immunoassay and by a conventional photometric assay.

Aspirin↗
PubMed1999

Salicylate promotes myeloperoxidase-initiated LDL oxidation: antagonization by its metabolite gentisic acid.

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. Tyrosyl radicals generated by myeloperoxidase (MPO) can act as prooxidants of LDL oxidation. Taking into consideration, that monophenolic compounds are able to form phenoxyl radicals in presence of peroxidases, we have tested salicylate, in its ability to act as a prooxidant in the MPO system. Measurement of conjugated dienes and lipid hydroperoxides were taken as indicators of lipid oxidation. Exposure of LDL preparations to MPO in presence of salicylate revealed that the drug could act as a catalyst of lipid oxidation in LDL. The radical scavenger ascorbic acid as well as heme poisons (cyanide, azide) and catalase were inhibitory. The main metabolite of salicylic acid, gentisic acid, showed inhibitory action in the MPO system. Even when lipid oxidation was maximally stimulated by salicylate the LDL oxidation was efficaciously counteracted in presence of gentisic acid at salicylate/gentisic acid ratios that could be reached in plasma of patients receiving aspirin medication. Gentisic acid was also able to impair the tyrosyl radical catalyzed LDL peroxidation. The results suggest that salicylate could act like tyrosine via a phenoxyl radical as a catalyst of LDL oxidative modification by MPO. But the prooxidant activity of this radical species is effectively counteracted by the salicylate metabolite gentisic acid.

Arteriosclerosis↗
PubMed1971

The enzymic degradation of alkyl-substituted gentisates, maleates and malates.

1. Cell-free extracts, prepared from a non-fluorescent Pseudomonas grown on m-cresol, oxidized gentisate and certain alkyl-substituted gentisates with the consumption of 1 mol of oxygen and the formation of 1 mol of pyruvate from 1 mol of substrate. 2. In addition to pyruvate, malate was formed from gentisate; citramalate was formed from 3-methylgentisate and 4-methylgentisate; 2,3-dimethylmalate was formed from 3,4-dimethylgentisate. 3. One enantiomer, d-(-)-citramalate, was formed enzymically from 3-methylgentisate, 4-methylgentisate and citraconate. l-(+)-Citramalate was formed from mesaconate by the same extracts. When examined as its dimethyl ester by gas-liquid chromatography, enzymically formed 2,3-dimethylmalate showed the same behaviour as one of the two racemates prepared from the synthetic compound. 4. Maleate, citraconate and 2,3-dimethylmaleate were rapidly hydrated by cell extracts, but ethylfumarate and 2,3-dimethylfumarate were not attacked. 5. Cell extracts oxidized 1,4-dihydroxy-2-naphthoate to give pyruvate and phthalate. 6. Alkylgentisates were oxidized by a gentisate oxygenase (EC 1.13.1.4) present in Pseudomonas 2,5. The ring-fission products were attacked by maleylpyruvase, but not by fumarylpyruvase, and their u.v.-absorption spectra were those expected for alkyl-substituted maleylpyruvates. 7. When supplemented with ATP, CoA, succinate and Mg(2+) ions, an enzyme system from cells grown with 2,5-xylenol formed pyruvate from d- but not from l-citramalate. Extracts from cells grown with dl-citramalate or with itaconate attacked both d- and l-citramalate; other alkylmalates were cleaved in similar fashion to give pyruvate or 2-oxobutyrate. 8. These results accord with a general sequence of reactions in which the benzene nucleus of an alkylgentisate is cleaved to give an alkyl-substituted maleylpyruvate. The ring-fission products are hydrolysed to give pyruvate, plus alkylmalic acids which then undergo aldol fissions, probably as their CoA esters. In Pseudomonas 2,5 several homologous sequences of this general type appear to be catalysed by a single battery of enzymes with broad substrate specificities, whereas the metabolic capabilities of the fluorescent Pseudomonas 3,5 are more restricted. 9. Intact cells of both organisms metabolize d-malic acid by reactions that have not been elucidated, but are different from those which degrade alkylmalates.

Alkylation↗