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PubMed2000

[Effects of 4-hydroxybenzoic acid methyl ester on the Ca(2+)-related functions of skinned skeletal muscle fibers from the guinea pig].

The amide-linked local anesthetics can be used safely in patients susceptible to malignant hyperthermia (MH). 4-Hydroxybenzoic acid methyl ester contained as a preservative in local anesthetic solution was suspected to have triggered MH in a patient who had received nerve block with lidocaine solution. The author investigated the effects of 4-hydroxybenzoic acid methyl ester on Ca(2+)-related functions of the skeletal muscle using saponin-treated skinned fibers from the extensor digitorum longus muscle of guinea pigs. 4-Hydroxybenzoic acid methyl ester accelerated the Ca2+ induced Ca2+ release (CICR) rate from the sarcoplasmic reticulum (SR) in a dose-dependent manner, and the Ca2+ concentration-CICR rate curves were shifted to the left with 4-hydroxybenzoic acid methyl ester at concentrations above 30 microM. 30 microM of 4-hydroxybenzoic acid methyl ester is associated with a plasma concentration of lidocaine intoxication which induces general convulsion. Ca2+ uptake, initial rate of Ca2+ uptake by the SR and Ca2+ sensitivity of the contractile system were not affected by 100 microM of 4-hydroxybenzoic acid methyl ester. These results suggest that amide-type local anesthetic solutions and agents containing 4-hydroxybenzoic acid methyl ester as a preservative may trigger MH in MH susceptible patients, if they are accidentally administered into the blood vessel.

Anesthetics, Local↗
PubMed2001

Intestinal bioavailability and biotransformation of 3-hydroxybenzo(a)pyrene in an isolated perfused preparation from channel catfish, Ictalurus punctatus.

The intestinal bioavailability and biotransformation of 3-hydroxybenzo(a)pyrene, a major metabolite of benzo(a)pyrene in many animal species, was investigated in an in situ isolated intestinal preparation from the channel catfish, and in vitro with preparations of catfish intestine and blood. 3-Hydroxybenzo(a)pyrene was a good substrate for adenosine 3'-phosphate 5'-phosphosulfate (PAPS)-sulfotransferase and UDP-glucuronosyltransferase in cytosol or microsomes prepared from intestinal mucosa. The benzo(a)pyrene-3-glucuronide and 3-sulfate conjugates were only very slowly hydrolyzed by intestinal beta-glucuronidase and sulfatase. The K(m) values for PAPS-sulfotransferase and UDP-glucuronosyltransferase were 0.4 and 1 microM, respectively, and V(max) were 1.61 +/- 1.08 nmol benzo(a)pyrene-3-sulfate/min/mg of cytosolic protein and 1.08 +/- 0.54 nmol benzo(a)pyrene-3-glucuronide/min/mg of microsomal protein. Hydrolytic enzyme activities were three orders of magnitude slower. In the in situ intestinal preparation, [(3)H]3-hydroxybenzo(a)pyrene was readily metabolized to the glucuronide and sulfate conjugates. After 1 h of incubation of 2 or 20 microM [(3)H]3-hydroxybenzo(a)pyrene in the in situ preparation, the luminal contents contained 3-hydroxybenzo(a)pyrene, benzo(a)pyrene-3,6-dione, benzo(a)pyrene-3-sulfate, and benzo(a)pyrene-3-glucuronide. Mucosal samples contained these components, as well as some unextractable material. The blood contained mainly benzo(a)pyrene-3-sulfate and an as yet unidentified metabolite of 3-hydroxybenzo(a)pyrene bound to hemoglobin. Some, but not all, blood samples contained small amounts of 3-hydroxybenzo(a)pyrene, benzo(a)pyrene-3-glucuronide, and benzo(a)pyrene-3,6-dione. These studies demonstrate the rapid phase 2 conjugation of a phenolic benzo(a)pyrene metabolite in intestinal mucosa, and the transfer of the phase 2 sulfate and glucuronide conjugates to blood.

Animals↗
PubMed1995

3-Hexaprenyl-4-hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis in Saccharomyces cerevisiae.

The biosynthesis of ubiquinone (coenzyme Q) was studied in Saccharomyces cerevisiae. Lipid extracts were prepared from wild-type yeast grown in the presence of p-[U-14C]- and p-[carboxy-14C]hydroxybenzoic acid. Ergosterol was removed by adsorption to digitonin-celite, and radiolabeled lipids were purified by sequential reverse-phase and normal-phase HPLC steps. Radiolabeled peaks were identified by comparison with synthetic standards using retention time and electron ionization mass spectrometric criteria. The recovery and identification of the unstable 3-hexaprenyl-4-hydroxybenzoic acid molecule were facilitated by treatment of the lipid extract with diazomethane under conditions that resulted in the formation of the stable derivatives methyl 3-hexaprenyl-4-hydroxybenzoate or methyl 3-hexaprenyl-4-methoxybenzoate. In stationary-phase yeast cultures, the major radioactive lipid products are coenzyme Q and 3-hexaprenyl-4-hydroxybenzoic acid, constituting 62 and 38% of the radioactive lipids, respectively. However, under log-phase growth conditions the biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid predominates (accounting for 81% of the radioactive lipids). The data indicate that in wild-type yeast, 3-hexaprenyl-4-hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis and that in log-phase growth this ubiquinone intermediate is present at fourfold higher abundance than the end product. The physiological rationale for this high concentration of a membrane-bound intermediate is unclear.

Benzoates↗
PubMed1985

Purification and properties of NADH/NADPH-dependent p-hydroxybenzoate hydroxylase from Corynebacterium cyclohexanicum.

Crude soluble extracts of Corynebacterium cyclohexanicum, grown on cyclohexanecarboxylic acid, were found to contain 4-hydroxybenzoate 3-hydroxylase which functions with NADH as well as NADPH. The purified enzyme preparation was electrophoretically homogeneous and contained FAD as prosthetic group. The relative molecular mass of the enzyme was estimated to be about 47000 by native and denaturated acrylamide gel electrophoresis, indicating that it is monomeric. The enzyme was stable at 60 degrees C for 10 min. The enzyme was highly specific for p-hydroxybenzoate. The activity was inhibited by several aromatic analogues of p-hydroxybenzoate such as p-aminobenzoate, p-fluorobenzoate, o-hydroxybenzoate, m-hydroxybenzoate, 2,4-dihydroxygenzoate, and 2,5-dihydroxybenzoate. The Km value for NADH was fairly constant, about 45 microM, in the pH range 7.0-8.4, whereas the Km value for NADPH increased from 63 microM to 170 microM as the pH rose from 7.0 to 8.4. V values in the same pH range, however, were approximately constant in both cases; about 30 mumol min-1 mg-1 for NADH, and 26 mumol min-1 mg-1 for NADPH. Mg2+ was required for full activity of the enzyme in low concentrations of phosphate buffer. The enzyme was inhibited by C1- which was non-competitive with respect to NADH, NADPH and p-hydroxybenzoate.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2004

A novel coupled enzyme assay reveals an enzyme responsible for the deamination of a chemically unstable intermediate in the metabolic pathway of 4-amino-3-hydroxybenzoic acid in Bordetella sp. strain 10d.

2-amino-5-carboxymuconic 6-semialdehyde is an unstable intermediate in the meta-cleavage pathway of 4-amino-3-hydroxybenzoic acid in Bordetella sp. strain 10d. In vitro, this compound is nonenzymatically converted to 2,5-pyridinedicarboxylic acid. Crude extracts of strain 10d grown on 4-amino-3-hydroxybenzoic acid converted 2-amino-5-carboxymuconic 6-semialdehyde formed from 4-amino-3-hydroxybenzoic acid by the first enzyme in the pathway, 4-amino-3-hydroxybenzoate 2,3-dioxygenase, to a yellow compound (epsilonmax = 375 nm). The enzyme in the crude extract carrying out the next step was purified to homogeneity. The yellow compound formed from 4-amino-3-hydroxybenzoic acid by this purified enzyme and purified 4-amino-3-hydroxybenzoate 2,3-dioxygenase in a coupled assay was identified as 2-hydroxymuconic 6-semialdehyde by GC-MS analysis. A mechanism for the formation of 2-hydroxymuconic 6-semialdehyde via enzymatic deamination and nonenzymatic decarboxylation is proposed based on results of spectrophotometric analyses. The purified enzyme, designated 2-amino-5-carboxymuconic 6-semialdehyde deaminase, is a new type of deaminase that differs from the 2-aminomuconate deaminases reported previously in that it primarily and specifically attacks 2-amino-5-carboxymuconic 6-semialdehyde. The deamination step in the proposed pathway differs from that in the pathways for 2-aminophenol and its derivatives.

4-Aminobenzoic Acid↗
PubMed2006

Metabolism of 4-amino-3-hydroxybenzoic acid by Bordetella sp. strain 10d: A different modified meta-cleavage pathway for 2-aminophenols.

Bordetella sp. strain 10d metabolizes 4-amino-3-hydroxybenzoic acid via 2-hydroxymuconic 6-semialdehyde. Cell extracts from 4-amino-3-hydroxybenzoate-grown cells showed high NAD(+)-dependent 2-hydroxymuconic 6-semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase activities, but no 2-hydroxymuconic 6-semialdehyde hydrolase activity. These enzymes involved in 4-amino-3-hydroxybenzoate metabolism were purified and characterized. When 2-hydroxymuconic 6-semialdehyde was used as substrate in a reaction mixture containing NAD(+) and cell extracts from 4-amino-3-hydroxybenzoate-grown cells, 4-oxalocrotonic acid, 2-oxopent-4-enoic acid, and 4-hydroxy-2-oxovaleric acid were identified as intermediates, and pyruvic acid was identified as the final product. A complete pathway for the metabolism of 4-amino-3-hydroxybenzoic acid in strain 10d is proposed. Strain 10d metabolized 2-hydroxymuconic 6-semialdehyde derived from 4-amino-3-hydroxybenzoic acid via a dehydrogenative route, not via a hydrolytic route. This proposed metabolic pathway differs considerably from the modified meta-cleavage pathway of 2-aminophenol and those previously reported for methyl- and chloro-derivatives.

4-Aminobenzoic Acid↗
PubMed2001

Microbial synthesis of p-hydroxybenzoic acid from glucose.

A series of recombinant Escherichia coli strains have been constructed and evaluated for their ability to synthesize p-hydroxybenzoic acid from glucose under fed-batch fermentor conditions. The maximum concentration of p-hydroxybenzoic acid synthesized was 12 g/L and corresponded to a yield of 13% (mol/mol). Synthesis of p-hydroxybenzoic acid began with direction of increased carbon flow into the common pathway of aromatic amino acid biosynthesis. This was accomplished in all constructs with overexpression of a feedback-insensitive isozyme of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase. Expression levels of enzymes in the common pathway of aromatic amino acid biosynthesis were also increased in all constructs to deliver increased carbon flow from the beginning to the end of the common pathway. A previously unreported inhibition of 3-dehydroquinate synthase by L-tyrosine was discovered to be a significant impediment to the flow of carbon through the common pathway. Chorismic acid, the last metabolite of the common pathway, was converted into p-hydroxybenzoic acid by ubiC-encoded chorismate lyase. Constructs differed in the strategy used for overexpression of chorismate lyase and also differed as to whether mutations were present in the host E. coli to inactivate other chorismate-utilizing enzymes. Use of overexpressed chorismate lyase to increase the rate of chorismic acid aromatization was mitigated by attendant decreases in the specific activity of DAHP synthase and feedback inhibition caused by p-hydroxybenzoic acid. The toxicity of p-hydroxybenzoic acid towards E. coli metabolism and growth was also evaluated.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1994

Predominance of glucuronidation over sulfation in metabolism of 1-hydroxybenzo[a]pyrene by isolated rat hepatocytes.

This study shows that 1-hydroxybenzo[a]pyrene glucuronide and 1-hydroxybenzo[a]pyrene sulfate are formed in isolated rat hepatocytes. Formation of these conjugates by hepatocytes incubated with 1-acetoxy-[G-3H]benzo[a]pyrene (100 microM) as a source of intracellular 1-hydroxy-[G-3H]benzo[a]pyrene was documented by comparison of the spectra of metabolites separated by HPLC with the spectra of 1-hydroxybenzo[a]pyrene glucuronide and 1-hydroxybenzo[a]pyrene sulfate standards. The rates of 1-hydroxybenzo[a]pyrene glucuronidation and sulfation were 7.72 +/- 1.03 and 0.68 +/- 0.02 nmol x mg dry wt.-1 x 30 min-1, respectively. The rate of 1-hydroxybenzo[a]pyrene glucuronide production by intact cells corresponded well with the total activity of UDP-glucuronosyltransferase(s) determined in permeabilized hepatocytes. Cryopreserved hepatocytes fully retained a high capacity to glucuronidate the benzo[a]pyrene phenol.

Animals↗
PubMed2001

Selective and quantitative analysis of 4-hydroxybenzoate preservatives by microemulsion electrokinetic chromatography.

A microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of 4-hydroxybenzoates and their impurities. These materials are commonly known as parabens and are widely used as preservatives in foods, cosmetics and pharmaceuticals. The method was shown to be selective and quantitative for the methyl, ethyl, propyl and butyl esters of 4-hydroxybenzoic acid. An internal standard, 4-hydroxyacetophenone, was employed to improve injection precision and detector linearity. In addition, 4-hydroxybenzoic acid, the major degradent, could also be monitored at the 0.1% (m/m) level. The method was successfully validated for assay and detection of the impurities in 4-hydroxybenzoic acid methyl ester and 4-hydroxybenzoic acid propyl ester samples and for the determination of 4-hydroxybenzoic acid methyl ester in a liquid pharmaceutical formulation. The determination of paraben content by MEEKC in a liquid sample was consistent with HPLC analysis. This work is the first reported validated MEEKC method and shows that the methodology can be successfully implemented into routine quality control testing.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed1989

Influence of alternate electron acceptors on the metabolic fate of hydroxybenzoate isomers in anoxic aquifer slurries.

The biodegradation of hydroxybenzoate isomers was investigated with samples obtained from two sites within a shallow anoxic aquifer. The metabolic fates of the substrates were compared in denitrifying, sulfate-reducing, and methanogenic incubations. Under the latter two conditions, phenol was detected as a major intermediate of p-hydroxybenzoate, but no metabolites were initially found with m- or o-hydroxybenzoate. However, benzoate accumulation was noted when metabolic inhibitors were used with these samples. About 9 to 17 days was required for >95% removal of the parent isomers under these conditions. When aquifer slurries were amended with nitrate, the equivalent removal of the hydroxybenzoates occurred within 4 days. In the denitrifying incubations, phenol was formed from all three hydroxybenzoates and accounted for about 30% of the initial substrate amendment. No benzoate was measured in these samples. All metabolites were identified by chromatographic mobility, mass spectral profiles, or both. Autoclaved controls were uniformly incapable of transforming the parent substrates. These results suggest that the anaerobic fate of hydroxybenzoate isomers depends on the relative substitution pattern and the prevailing ecological conditions. Furthermore, since these compounds are central metabolites formed during the breakdown of many aromatic chemicals, our findings may help provide guidelines for the reliable extrapolation of metabolic fate information from diverse anaerobic environments.

Journal Article↗
PubMed1972

Biochemical and genetic studies on ubiquinone biosynthesis in Escherichia coli K-12:4-hydroxybenzoate octaprenyltransferase.

Three ubiquinone-deficient mutants of Escherichia coli unable to convert 4-hydroxybenzoate into 3-octaprenyl-4-hydroxybenzoate were isolated and examined. The results of genetic analysis suggest that each of the mutants carries a mutation in a gene designated ubiA which can be represented at minute 79 on the E. coli chromosome map. The conversion of 4-hydroxybenzoate into 3-octaprenyl-4-hydroxybenzoate, catalyzed by 4-hydroxybenzoate octaprenyltransferase, was studied with a strain of E. coli that is blocked in the common pathway of aromatic biosynthesis and consequently accumulates the precursor of the side chain of ubiquinone. Both the side-chain precursor and 4-hydroxybenzoate octaprenyltransferase were shown to be membrane-bound. The enzyme required Mg(2+) for optimal activity. The ubiA(-) mutants were found to lack 4-hydroxybenozate octaprenyltransferase activity, which suggested that the ubiA gene is the structural gene coding for this enzyme.

Alkyl and Aryl Transferases↗
PubMed1992

Anaerobic growth of Rhodopseudomonas palustris on 4-hydroxybenzoate is dependent on AadR, a member of the cyclic AMP receptor protein family of transcriptional regulators.

The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris converts structurally diverse aromatic carboxylic acids, including lignin monomers, to benzoate and 4-hydroxybenzoate under anaerobic conditions. These compounds are then further degraded via aromatic ring-fission pathways. A gene termed aadR, for anaerobic aromatic degradation regulator, was identified by complementation of mutants unable to grow anaerobically on 4-hydroxybenzoate. The deduced amino acid sequence of the aadR product is similar to a family of transcriptional regulators which includes Escherichia coli Fnr and Crp, Pseudomonas aeruginosa Anr, and rhizobial FixK and FixK-like proteins. A mutant with a deletion in aadR failed to grow on 4-hydroxybenzoate under anaerobic conditions and grew very slowly on benzoate. It also did not express aromatic acid-coenzyme A ligase II, an enzyme that catalyzes the first step of 4-hydroxybenzoate degradation, and it was defective in 4-hydroxybenzoate-induced expression of benzoate-coenzyme A ligase. The aadR deletion mutant was unaffected in other aspects of anaerobic growth. It grew normally on nonaromatic carbon sources and also under nitrogen-fixing conditions. In addition, aerobic growth on 4-hydroxybenzoate was indistinguishable from that of the wild type. These results indicate that AadR functions as a transcriptional activator of anaerobic aromatic acid degradation.

Amino Acid Sequence↗
PubMed1986

Binding of 2-hydroxybenzo(a)pyrene to estrogen receptors in rat cytosol.

The potent carcinogen 2-hydroxybenzo(a)pyrene (2-OH-BP) competes for binding to the estrogen receptor in the cytosol of rat uterus and liver. The dissociation constant (K1) for this interaction is congruent to 2 X 10(-5) M. In contrast, 4-hydroxybenzo(a)pyrene does not bind to the estrogen receptor; 1-hydroxybenzo(a)pyrene, 5-hydroxybenzo(a)pyrene, 6-hydroxybenzo(a)pyrene, and 12-hydroxybenzo(a)pyrene bind less tightly than does 2-OH-BP. These five chemicals are not carcinogenic. We suggest that the estrogen receptor may mediate the carcinogenic effect of 2-OH-BP or of related chemicals. One possibility is that the receptor might convey 2-OH-BP to specific sites in DNA.

Animals↗
PubMed2001

Analysis of the pobA and pobR genes controlling expression of p-hydroxybenzoate hydroxylase in Azotobacter chroococcum.

We report the cloning and analysis of a gene and its cognate regulatory element from a member of the Azotobacteriaceae which are involved in the breakdown of an aromatic compound. The genes from Azotobacter chroococcum encoding p-hydroxybenzoate hydroxylase (pobA) and its regulatory protein (pobR) were cloned from a genomic library and sequenced. Sequence analysis of pobA revealed homology with other bacterial p-hydroxybenzoate hydroxylase enzymes. Residues essential to the structure and function of the enzyme have been conserved. The pobR gene encodes a DNA binding regulatory protein with similarity to proteins from the AraC/XylS family of transcriptional activators. A fragment containing both pobA and pobR was cloned into pUC19 and p-hydroxybenzoate hydroxylase activity was induced in Escherichia coli by the addition of p-hydroxybenzoate. A frame-shift mutation introduced into the pobR gene prevented expression of p-hydroxybenzoate hydroxylase, indicating that PobR is the protein required for transcription of pobA. Interestingly, A. chroococcum PobR has no homology to the PobR protein that is the transcriptional activator of pobA in Acinetobacter strain ADP1, a protein that is homologous to the IclR family of transcriptional regulators. However, PobR from A. chroococcum is homologous to several other proteins, suggesting that these proteins will also function as transcriptional activators of pobA.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2004

Increased positive electrostatic potential in p-hydroxybenzoate hydroxylase accelerates hydroxylation but slows turnover.

Para-hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the isoalloxazine ring within the protein structure. In this paper, we examine the effect of increased positive electrostatic potential in the active site upon the catalytic process with the enzyme mutation, Glu49Gln. This mutation removes a negative charge from a conserved buried charge pair. The properties of the Glu49Gln mutant enzyme are consistent with increased positive potential in the active site, but the mutant enzyme is difficult to study because it is unstable. There are two important changes in the catalytic function of the mutant enzyme as compared to the wild-type. First, the rate of hydroxylation of p-hydroxybenzoate by the transiently formed flavin hydroperoxide is an order of magnitude faster than in the wild-type. This result is consistent with one function proposed for the positive potential in the active site-to stabilize the negative C-4a-flavin alkoxide leaving group upon heterolytic fission of the peroxide bond. However, the mutant enzyme is a poorer catalyst than the wild-type enzyme because (unlike wild-type) the binding of p-hydroxybenzoate is a rate-limiting process. Our analysis shows that the mutant enzyme is slow to interconvert between conformations required to bind and release substrate. We conclude that the new open structure found in crystals of the Arg220Gln mutant enzyme [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613] is integral to the process of binding and release of substrate from oxidized enzyme during catalysis.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2004

Oxygen reactions in p-hydroxybenzoate hydroxylase utilize the H-bond network during catalysis.

para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyses a reaction in two parts: reduction of the flavin adenine dinucleotide (FAD) in the enzyme by reduced nicotinamide adenine dinucleotide phosphate (NADPH) in response to binding p-hydroxybenzoate to the enzyme and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the protein and isoalloxazine ring during catalysis. Earlier research showed that reduction of FAD occurs when the isoalloxazine of the FAD moves to the surface of the protein to allow hydride transfer from NADPH. This move is coordinated with protein rearrangements that are triggered by deprotonation of buried p-hydroxybenzoate through a H-bond network that leads to the surface of the protein. In this paper, we examine the involvement of this same H-bond network in the oxygen reactions-the initial formation of a flavin-C4a-hydroperoxide from the reaction between oxygen and reduced flavin, the electrophilic attack of the hydroperoxide upon the substrate to form product, and the elimination of water from the flavin-C4a-hydroxide to form oxidized enzyme in association with product release. These reactions were measured through absorbance and fluorescence changes in the FAD during the reactions. Results were collected over a range of pH for the reactions of wild-type enzyme and a series of mutant enzymes with the natural substrate and substrate analogues. We discovered that the rate of formation of the flavin hydroperoxide is not influenced by pH change, which indicates that the proton required for this reaction does not come from the H-bond network. The rate of the hydroxylation reaction increases with pH in a manner consistent with a pK(a) of 7.1. We conclude that the H-bond network abstracts the phenolic proton from p-hydroxybenzoate in the transition state of oxygen transfer. The rate of formation of oxidized enzyme increases with pH in a manner consistent with a pK(a) of 7.1, indicating the involvement of the H-bond network. We conclude that product deprotonation enhances the rate of a specific conformational change required for both product release and the elimination of water from C4a-OH-FAD.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1998

Lys42 and Ser42 variants of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens reveal that Arg42 is essential for NADPH binding.

The conserved Arg42 of the flavoprotein p-hydroxybenzoate hydroxylase is located at the entrance of the active site in a loop between helix H2 and sheet E1 of the FAD-binding domain. Replacement of Arg42 by Lys or Ser decreases the turnover rate of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by more than two orders of magnitude. Rapid reaction kinetics show that the low activity of the Arg42 variants results from impaired binding of NADPH. In contrast to an earlier conclusion drawn for p-hydroxybenzoate hydroxylase from Acinetobacter calcoaceticus, substitution of Arg42 with Ser42 in the enzyme from P. fluorescens hardly disturbs the binding of FAD. Crystals of [Lys42]p-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of the Lys42 variant is virtually indistinguishable from the native enzyme with the flavin ring occupying the interior position within the active site. Lys42 in the mutant structure interacts indirectly via a solvent molecule with the 3-OH of the adenosine ribose moiety of FAD. Substrate perturbation difference spectra suggest that the Arg42 replacements influence the solvent accessibility of the flavin ring in the oxidized enzyme. In spite of this, the Arg42 variants fully couple enzyme reduction to substrate hydroxylation. Sequence-comparison studies suggest that Arg42 is involved in binding of the 2'-phosphoadenosine moiety of NADPH.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2004

Characteristics of p-hydroxybenzoic acid oxidation using Fenton's reagent.

The objective of this work was to investigate the oxidation of p-hydroxybenzoic acid by the Fenton's reagent. Batch experiments were carried out to investigate the influence of the most important process variables: hydrogen peroxide and ferrous salt concentrations, pH, and temperature. The optimal results obtained indicate that p-hydroxybenzoic acid can be effectively degraded using Fenton's oxidation within 10-min reaction time by using a molar ratio H2O2: p-hydroxybenzoic acid of 4:1 and H2O2:Fe2+ of 15:1, at 30 degrees C and pH=3.0. The behavior of ORP and pH along the reaction time for different values of R (molar ratio H2O2: p-hydroxybenzoic acid) was also discussed. A pseudo-first order model was applied to describe the oxidation kinetics of p-hydroxybenzoic acid by Fenton's reagent.

Hydrogen Peroxide↗