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PubMed1991

NMR studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C- and 15N-enriched FAD. The protein preparations were studied by 13C-NMR, 15N-NMR and 31P-NMR techniques in the oxidized and in the two-electron-reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the pi electron distribution in oxidized free p-hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e.g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an alpha-helix microdipole. In the reduced state, the flavin in p-hydroxybenzoate hydroxylase is bound in the anionic form, i.e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, C10a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state. The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4 alpha. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2-hybridized N5 atom and a slightly sp3-hybridized N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1992

Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed mutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214-->Ala and Arg214-->Gln, the effector role of substrate is lost. For enzyme Arg214-->Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214-->Lys fails to stabilize the 4 alpha-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214-->Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1980

Polyprenyl p-hydroxybenzoate carboxylase in flagellation of Salmonella typhimurium.

Flagellation of Salmonella typhimurium was found to require a functional pathway for ubiquinone biosynthesis as well as growth in the presence of appropriate carboxylic acids. Induction of flagellation by carboxylic acids was shown to induce incorporation of p-hydroxybenzoic acid into polyprenylphenol. Constitutive flagellation was found to correlate with constitutive incorporation of p-hydroxybenzoic acid into polyprenylphenol. A novel pathway for polyprenyl p-hydroxybenzoic acid decarboxylation to polyprenylphenol was implicated in flagellation of S. typhimurium.

Biological Transport↗
PubMed1993

Identification of the transcriptional activator pobR and characterization of its role in the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase in Acinetobacter calcoaceticus.

We have identified pobR, a gene encoding a transcriptional activator that regulates expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase (PobA) in Acinetobacter calcoaceticus ADP1. Inducible expression of cloned pobA in Escherichia coli depended upon the presence of a functional pobR gene, and mutations within pobR prevented pobA expression in A. calcoaceticus. A pobA-lacZ operon fusion was used to demonstrate that pobA expression in A. calcoaceticus is enhanced up to 400-fold by the inducer p-hydroxybenzoate. Inducer concentrations as low as 10(-7) M were sufficient to elicit partial induction. Some structurally related analogs of p-hydroxybenzoate, unable to cause induction by themselves, were effective anti-inducers. The nucleotide sequence of pobR was determined, and the activator gene was shown to be transcribed divergently from pobA; the genes are separated by 134 DNA base pairs. The deduced amino acid sequence yielded a polypeptide of M(r) = 30,764. Analysis of this sequence revealed at the NH2 terminus a stretch of residues with high potential for forming a helix-turn-helix structure that could serve as a DNA-binding domain. A conservative amino acid substitution (Arg-61-->His-61) in this region inactivated PobR. The primary structure of PobR appears to be evolutionarily distinct from the four major families of NH2-terminal helix-turn-helix containing bacterial regulatory proteins that have been identified thus far.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1979

Assimilation of protocatechuic acid and p-hydroxybenzoic acid as an aid to laboratory identification of Candida parapsilosis and other medically important yeasts.

Test for the ability of yeasts isolated from clinical specimens to utilize protocatechuic acid and p-hydroxybenzoic acid were carried out by using techniques that are commonly employed to test assimilation of carbon sources. A total of 60 isolates of Candida parapsilosis and 5 isolates of Candida humicola readily assimilated these two phenolic acids, whereas other Candida species gave uniformly negative results. Cryptococcus albidus, Cryptococcus terreus, and some isolates of Cryptococcus laurentii also assimilated protocatechuate and p-hydroxybenzoate, whereas Cryptococcus neoformans did not. Results of these tests suggest that assimilation of protocatechuate and p-hydroxybenzoate may be a useful characteristic, when used in conjunction with traditional tests, for identifying C. parapsilosis and C. albidus.

Candida↗
PubMed2001

[Research on the separation of three isomers of hydroxybenzoic acid by capillary zone electrophoresis].

The separation behavior of o-hydroxybenzoic acid, m-hydroxybenzoic acid and p-hydroxybenzoic acid in high performance capillary zone electrophoresis was investigated using cetyltrimethylammonium bromide (CTAB) as reversed reagent for electroosmotic flow. The effects of pH of buffer solution and volume fraction of methanol on the separation, peak shape and elution order were studied.

Buffers↗
PubMed2002

[Effect of vanillin and P-hydroxybenzoic acid on physiological characteristics of Chinese fir seedlings].

Effects of vanillin and P-hydroxybenzoic acid at different concentrations on physiological characteristic of Chinese fir seedlings were studied by potted experiment. The results showed that 10 mmol.L-1 and 1 mmol.L-1 of two kinds of phenolics significantly reduced the content of chlorophyll, rate of photosynthesis and root activity, and that the higher the concentration of vanillin and P-hydroxybenzoic acid, the more the physiological activities was inhibited. By treatment with 1 and 10 mmol.L-1 vanillin, Chinese fir seedlings reduced its photosynthesis rate 25.1% and 37.0%, transpiration rate 20.3% and 37.0%, stomata conductance 33.7% and 46.8% and root activity 51.6% and 78.8%, respectively. The results suggested that vanillin and P-hydroxybenzoic acid accumulated in the soil by continuous cropping of Chinese fir may have some allelopathic effect on the seedlings of Chinese fir and the effect is one of the factors leading to the low productivity of continuously cropped Chinese fir forest.

Antioxidants↗
PubMed2004

Characterization of protocatechuate 4,5-dioxygenase induced from p-hydroxybenzoate-cultured Pseudomonas sp. K82.

Pseudomonas sp. K82 has been reported to be an aniline-assimilating soil bacterium. However, this strain can use not only aniline as a sole carbon and energy source, but can also utilize benzoate, p-hydroxybenzoate, and aniline analogues. The strain accomplishes this metabolic diversity by using different aerobic pathways. Pseudomonas sp. K82, when cultured in p-hydroxybenzoate, showed extradiol cleavage activity of protocatechuate. In accordance with those findings, our study attempted the purification of protocatechuate 4,5-dioxygenase (PCD 4,5). However the purified PCD 4,5 was found to be very unstable during purification. After Q-sepharose chromatography was performed, the crude enzyme activity was augmented by a factor of approximately 4.7. From the Q-sepharose fraction which exhibited PCD 4,5 activity, two subunits of PCD4,5 (alpha subunit and beta subunit) were identified using the N-terminal amino acid sequences of 15 amino acid residues. These subunits were found to have more than 90% sequence homology with PmdA and PmdB of Comamonas testosteroni. The molecular weight of the native enzyme was estimated to be approximately 54 kDa, suggesting that PCD4,5 exists as a heterodimer (alpha1beta1). PCD 4,5 exhibits stringent substrate specificity for protocatechuate and its optimal activity occurs at pH 9 and 15 degrees C. PCR amplification of these two subunits of PCD4,5 revealed that the alpha subunit and beta subunit occurred in tandem. Our results suggest that Pseudomonas sp. K82 induced PCD 4,5 for the purpose of p-hydroxybenzoate degradation.

Amino Acid Sequence↗
PubMed2005

Metabolism of xenobiotics in the incubated hen's egg: investigations with ethyl 4-hydroxybenzoate.

It is well known that in vitro preparations of the chick embryo can functionalise and conjugate selected model substances. Based on this biochemical knowledge an ex vivo model was developed to study xenobiotic metabolism in the incubated hen's egg. The xenobiotic is injected into the yolk sac, i.e. the nutritional compartment, on day 6 and metabolites are identified in the excretion medium of the embryonic kidneys (allantoic fluid) on day 11. During this developmental period the embryo lacks or has very limited sensitivity. Thus the model is in accordance with the 3R concept. In the present investigation ethyl 4-hydroxybenzoate was chosen as a test substance. Concentrations of this paraben up to 24 mg/egg did not affect embryo viability. After inoculation of 6 mg/egg, 4-hydroxybenzoic acid and 4,4'-dihydroxy-L-ornithuric acid were identified in their free form. The 4-hydroxybenzoic acid was also eliminated in its conjugated form (glucuronide and/or sulphate). No unchanged paraben was excreted. 4,4'-dihydroxy-L-ornithuric acid [2,5-bis-(4-hydroxybenzoylamino)pentanoic acid] is a new metabolite. The structure of this amino acid conjugate was elucidated by synthesis and spectral methods (MS, 1H and 13C NMR).

Animal Testing Alternatives↗
PubMed1983

Reaction of 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase with hydrogen peroxide. Formation of a covalent flavin-protein linkage.

Hydrogen peroxide reacts with 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase to yield a long wavelength intermediate (lambda max = 360, 620 nm) which can be isolated in stable form on removal of excess H2O2. The blue flavin derivative slowly decays in a second peroxide-dependent reaction to yield a new flavin product lacking long wavelength absorbance (lambda max = 408, 472 nm). This final peroxide-modified enzyme binds p-hydroxybenzoate with a 10-fold lower affinity than does the native enzyme; furthermore, substrate binding leads to the inhibition of enzyme reduction by NADPH. Trichloroacetic acid treatment of the final peroxide-modified enzyme results in the quantitative conversion of the bound flavin to free FAD. However, gel filtration of the modified enzyme in guanidine hydrochloride at neutral pH leads to the co-elution of protein and modified flavin. The nondenatured peroxide product reacts rapidly with hydroxylamine to yield 2-NHOH-substituted FAD. These observations indicate that the secondary reaction of peroxide with the blue intermediate from 2-thio-FAD p-hydroxybenzoate hydroxylase results in the formation of an acid-labile covalent flavin-protein linkage within the enzyme active site, involving the flavin C-2 position.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1994

[13-week subchronic oral toxicity study of isopropyl p-hydroxybenzoate in F344 rats].

A 13-week subchronic oral toxicity study of isopropyl p-hydroxybenzoate was performed in both sexes of F344 rats. CRF-1 diet containing 0, 0.25, 1.25, 2.5 or 5% isopropyl p-hydroxybenzoate was fed to 5 randomly constituted groups of animals, each consisting of 10 males and 10 females, to determine appropriate dose levels for a subsequent 2-year carcinogenicity study. No animals died during the administration period. Significant suppression of body weight gain was observed in males of the 2.5% and 5% isopropyl p-hydroxybenzoate groups, and in females of the groups treated with 1.25% or above as compared with the control group. Serum biochemistry evaluations revealed increases in gamma-GTP and total cholesterol in male groups treated with 2.5% or more and increases in gamma-GTP, ALP and BUN in female groups treated with 1.25% or more, as compared to the controls. Histopathologically, centrilobular hepatocellular swelling was observed in males treated with 2.5% or more and in females of the 5% group. In the affected populations, hepatocytes filled with small vacuoles, possibly of lipid native, were sometimes found. An increased severity of intracytoplasmic eosinophilic globule formation in the renal proximal tubular epithelia of males of the 5% group was noted. Based on these results, a dietary concentration of 1% in males or 0.5% in females was concluded to be a suitable maximum tolerable dose (MTD) of this chemical for a 2-year carcinogenicity study in rats.

Administration, Oral↗
PubMed2005

Co-production of caffeic acid and p-hydroxybenzoic acid from p-coumaric acid by Streptomyces caeruleus MTCC 6638.

In a culture medium of Streptomyces caeruleus MTCC 6638 grown with p-coumaric acid (5 mM) as the sole source of carbon, co-production of caffeic acid and p-hydroxybenzoic acid was observed. Both caffeic acid and p-hydroxybenzoic acid are important phenolic compounds with pharmaceutical importance. These biotransformed products were identified by high-performance liquid chromatography and electrospray ionization mass spectrometry. Obtained data suggest that p-coumaric acid was possibly utilized by two different routes, resulting in the formation of a hydroxycinnamate and a hydroxybenzoate compound. However, higher concentration of p-coumaric acid (10 mM) favoured caffeic acid formation. Addition of 5 mM p-coumaric acid into S. caeruleus cultures pre-grown on minimal medium with 1.0 g/l glucose resulted in the production of 65 mg/l caffeic acid. Furthermore, S. caeruleus cells were able to produce the maximum amount of caffeic acid when pre-grown on nutrient broth for 16 h. Under this condition, the addition of 5 mM p-coumaric acid was sufficient for the S. caeruleus culture to produce 150 mg/l caffeic acid, with a molar yield of 16.6% after 96 h of incubation.

Caffeic Acids↗
PubMed2002

Isolation from a shea cake digester of a tannin-tolerant Escherichia coli strain decarboxylating p-hydroxybenzoic and vanillic acids.

A facultatively anaerobic, mesophilic, Gram-negative, non-motile, non-sporulated bacterium, designated strain C2, was isolated from an anaerobic digester fed with shea cake rich in tannins and aromatic compounds and previously inoculated with anaerobic sludge from the pit of a slaughterhouse, after enrichment on tannic acid. The straight rods occurred singly or in pairs. Strain C2 fermented numerous carbohydrates (fructose, galactose, glucose, lactose, mannose, maltose, melibiose, raffinose, rhamnose, ribose, saccharose, sorbitol, trehalose, and xylose) and peptides (Biotrypcase, Casamino acids, and yeast extract), producing acid and gas, and had a G + C content of 51.6 +/- 0.1 mol %. Strain C2 was very closely related to Escherichia coli (= DSM 30083(T)) phylogenetically (similarity of 99%), genotypically (DNA homology of 79%), and phenotypically. The isolate tolerated tannic acid (hydrolyzable tannin) and decarboxylated by non-oxidative decarboxylation only p-hydroxybenzoic and vanillic acids to their corresponding phenol and guaicol, under anaerobic and aerobic conditions without further degradation. Adding glucose increased growth and the rate of conversion. High concentrations of p-hydroxybenzoic acid or vanillic acid inhibited growth, and decarboxylation could not occur completely, suggesting phenol toxicity. In contrast, the type strain of E. coli cannot metabolize p-hydroxybenzoic and vanillic acids, anaerobically or aerobically, with or without glucose added.

Aerobiosis↗
PubMed1997

3-Hydroxybenzoate:coenzyme A ligase and 4-coumarate:coenzyme A ligase from cultured cells of Centaurium erythraea.

3-Hydroxybenzoate:coenzyme A ligase, an enzyme involved in xanthone biosynthesis, was detected in cell-free extracts from cultured cells of Centaurium erythraea Rafn. The enzyme was separated from 4-coumarate:coenzyme A ligase by fractionated ammonium sulphate precipitation and hydrophobic interaction chromatography. The CoA ligases exhibited different substrate specificities. 3-Hydroxybenzoate:coenzyme A ligase activated 3-hydroxybenzoic acid most efficiently and lacked affinity for cinnamic acids. In contrast, 4-coumarate:CoA ligase mainly catalyzed the activation of 4-coumaric acid but did not act on benzoic acids. The two enzymes were similar with respect to their relative molecular weight, their pH and temperature optima, their specific activity and the changes in their activity during cell culture growth.

Cells, Cultured↗
PubMed1991

Partial purification, properties, and kinetic studies of UDP-glucose:p-hydroxybenzoate glucosyltransferase from cell cultures of Lithospermum erythrorhizon.

A glucosyltransferase, which catalyzed the transfer of glucose from UDP-glucose (UDPG) to p-hydroxybenzoate (PHB) in cell cultures of Lithospermum erythrorhizon Sieb. et Zucc., Boraginaceae, was purified 219-fold by ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephadex G-150, and phenyl-Sepharose Cl-4B. p-Hydroxybenzoic acid O-beta-D-glucoside (PHB-glc) was identified as a product of the enzymatic reaction. This glucosyltransferase has a molecular weight of 47,500 Da, an isoelectric point at pH 5.0, and a pH optimum of 7.8. The enzyme does not sediment at 100,000g. Enzyme activity did not require metal cofactors. The enzyme was highly specific for p-hydroxybenzoate (Km 0.264 mM) and UDP-glucose (Km 0.268 mM). Initial velocity studies suggest that the enzyme reaction mechanism is a sequential rather than a ping-pong mechanism. Product inhibition patterns are consistent with an ordered sequential bi-bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP the final product released. The data indicate the formation of a dead-end complex between PHB-glc and the enzyme. Uncompetitive inhibition by the substrate PHB can be put down to the formation of an abortive complex between E-UDP and PHB.

Glucosyltransferases↗
PubMed1994

Modulation by alkyl p-hydroxybenzoates of voltage- and ligand-gated channels in peripheral neuronal cells.

Effects of alkyl p-hydroxybenzoates (APHBs), which are used as preservatives, on ion channels were investigated in rat pheochromocytoma PC12 cells. Methyl p-hydroxybenzoate (MPHB; 300 microM) and butyl p-hydroxybenzoate (BPHB; 300 microM) inhibited Ba2+ current passing through Ca2+ channels, and facilitated the inactivation of the Ba2+ current. K+ current obtained with a depolarizing voltage-step was also suppressed by 300 microM MPHB or 300 microM BPHB. The extent of the suppression of the K+ current was not affected by extracellular Cd2+, suggesting that the suppression of the K+ current is not a secondary effect arising from the Ca2+ channel inhibition. An inward current activated by acetylcholine (ACh; 100 microM) was abolished by 300 microM BPHB, and it was partially blocked by 300 microM MPHB. In contrast to the ACh-activated current, an inward current activated by ATP (30 microM) was markedly potentiated by 300 microM BPHB. The results suggest that APHBs exert significant effects on the voltage- and ligand-gated channels. The significance of these channel modifications were discussed in relation to reported effects of APHBs, including induction of minor irritation.

Acetylcholine↗