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PubMed2004

Surface-enhanced Raman scattering of a series of n-hydroxybenzoic acids (n = P, M and O) on the silver nano-particles.

Surface-enhanced Raman scattering (SERS) spectra of a series of n-hydroxybenzoic acids (n-HBA, n = P, M and O) adsorbed on the silver nano-particles were studied, respectively, in the silver colloidal solution and on the dried silver-coated filter paper. On the same substrate, the different molecules' SERS spectra were different, while on the different substrates the same molecules' SERS spectra were also different. Significant changes were found in the SERS spectra of PHBA molecules adsorbed on the two substrates, and the changes found in MHBA's spectra on two substrates were next to PHBA's, while almost no changes were found in the spectra of OHBA molecules. Moreover, it was found, on the filter paper, that the SERS spectra of the same molecules would change with the coverage density of the silver nano-particles. The analyses showed that the origins of these changes were the different adsorption behavior of molecules adsorbed on the silver nano-particles. Because in these three molecules the relative positions of the carboxyls and hydroxyls on the benzenes are different, the adsorption behaviors of these three molecules adsorbed on the silver surfaces are also different. The experimental results suggest that the surface characteristic of the substrate and the surface configuration of the adsorbate could exert a great influence on the adsorption behavior of the adsorbates on the substrates.

Adsorption↗
PubMed2005

The investigation of a series of n-hydroxybenzoic acids (n=p, m, o) on a new surface enhanced Raman scattering active substrate.

Ag nanoparticles, organized on indium tin oxide (ITO) surface, can act as a new surface enhanced Raman scattering (SERS) active substrate as well as a dry electrode. Compared with the traditional SERS-active substrate, its notable advantage is that the microcosmic changes of the film can be reflected by the SERS spectrum during the investigation of the film's electrical and other macroscopic characteristics. To illuminate the above-mentioned property of this new substrate, a series of n-hydroxybenzoic acids (n-HBA; n=p, m and o) was tested as probe molecules by SERS technique. These SERS spectra indicate that the significant changes of frequencies as well as intensities, respectively, arise from the changes of the adsorption behavior along with the proportional variation of molecules and silver nanoparticles. Excellent SERS signals prove that the silver nanoparticles-coated ITO is a highly SERS-active substrate and can efficiently reflect the microcosmic property of the film, which suggest it has promising potential of being a new technique for further application in the field of thin-film research.

Colloids↗
PubMed2004

High affinity for the rat brain sodium channel of newly discovered hydroxybenzoate saxitoxin analogues from the dinoflagellate Gymnodinium catenatum.

The paralytic shellfish poison family has been recently extended by the discovery of several analogues possessing a hydoxybenzoate moiety instead of the carbamoyl group one finds in saxitoxin, the parent molecule of this toxin family. We have investigated the potency of these new analogues on a representative isoform of the pharmacological target of these toxins, the voltage gated sodium channel. These toxins were found to have K1's in the low nanomolar range, only slightly less potent than saxitoxin. The hydroxybenzoate group may increase the lipophilicity of these toxins and improve their ability to pass through epithelia and therefore its uptake and elimination in both intoxication victims and animals that bioaccumulate paralytic shellfish toxins.

Animals↗
PubMed2000

Antioxidative galloyl esters as enzyme inhibitors of p-hydroxybenzoate hydroxylase.

Gallic acid and its esters were evaluated as enzyme inhibitors of recombinant p-hydroxybenzoate hydroxylase (PHBH), a NADPH-dependent flavin monooxygenase from Pseudomonas aeruginosa. n-Dodecyl gallate (DG) (IC(50)=16 microM) and (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50)=16 microM), a major component of green tea polyphenols, showed the most potent inhibition, while product-like gallic acid did not inhibit the enzyme significantly (IC(50)>250 microM). Inhibition kinetics revealed that both DG and EGCG inhibited PHBH in a non-competitive manner (K(I)=18.1 and 14.0 microM, respectively). The enzyme inhibition was caused by specific binding of the antioxidative gallate to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction. Molecular modeling predicted that EGCG binds to the enzyme in the proximity of the FAD binding site via formation of three hydrogen bonds.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1999

Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase. Implications for NADPH recognition and structural stability.

Phe161 and Arg166 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site-directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 A is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 A revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1987

High-performance liquid chromatographic determination of six p-hydroxybenzoic acid esters in cosmetics using Sep-Pak florisil cartridges for sample pre-treatment.

A rapid and simple method is described for the simultaneous determination of methyl, ethyl, isopropyl, n-propyl, isobutyl and n-butyl p-hydroxybenzoic acid esters (parabens) in cosmetics by high-performance liquid chromatography (HPLC). The method involves a single extraction of parabens with diethyl ether and clean-up on a Sep-Pak Florisil cartridge. Fat-soluble excipients in the diethyl ether extracts are removed through the cartridges with hexane-chloroform (75:25). Parabens are then eluted from the cartridges with hexane-ethyl acetate (70:30) and determined by HPLC on a reversed-phase column with water-methanol (50:50) as the mobile phase using sec.-butylpraben as an internal standard. The method was applied to samples with complicated matrices such as cream, milk lotion, lotion and cleansing foam, and the recoveries were 99.0-102.3% with coefficients of variation of 0.3-1.2%.

Chromatography, High Pressure Liquid↗
PubMed1983

Comparison of the three-dimensional protein and nucleotide structure of the FAD-binding domain of p-hydroxybenzoate hydroxylase with the FAD- as well as NADPH-binding domains of glutathione reductase.

The chain fold of the FAD-binding domain of p-hydroxybenzoate hydroxylase resembles the chain folds of the two nucleotide-binding domains of glutathione reductase. This fold consists of a four-stranded parallel beta-sheet sandwiched between a three-stranded antiparallel beta-sheet and alpha-helices. The nucleotides bind in similar positions relative to this chain fold. The best superposition of the folds has been established and geometrically quantified, giving rise to an equivalencing scheme for 110 residue positions, of which only four residues are identical in all three domains. It is discussed whether this chain fold is also present in a number of other FAD-binding proteins with known sequence. After the second strand of the parallel beta-sheet both FAD-binding domains contain long chain excursions, which make intimate contacts to rather distant parts of the respective molecules. In the environment of the isoalloxazine rings we observe interesting similarities. In both enzymes the si-face of this ring is covered by polypeptide, and only the re-face is accessible for the cofactor NADPH. Furthermore, there is a long alpha-helix in each enzyme, which points with its N-terminal start to the O-2 alpha region of isoalloxazine. These helices are spatially in the same position with respect to the isoalloxazine ring but are at quite different positions along the polypeptide chain. Since they can stabilize a negative charge around O-2 alpha, they may be important for the catalytic processes.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2002

Co-oxidation of p-hydroxybenzoic acid and atrazine by the Fenton's like system Fe(III)/H2O2.

The system Fe(III)/H2O2 has been used to oxidise an aqueous solution of p-hydroxybenzoic acid (pHB) in the absence of light. In the process, typical operating variables such as reagent concentration exert a positive influence in the pHB degradation rate. Optimum pH has been found to be around 3. The kinetic study suggests that the mechanism involved in this system differs to some extent from that reported for the classic Fenton's chemistry in pure water. Thus, formation of a complex Fe(III)-pHB seems to be a key step to initiate the oxidising mechanism. Stoichiometric measurements of the H2O2 consumption per mole of pHB degraded indicate a possible reduction of complexed Fe(III). Simultaneous oxidation of pHB (and other similar compounds such as tyrosol (Ty) or p-coumaric acid (pCu)) and atrazine have shown a synergistic effect of the first substance to remove the pesticide.

Atrazine↗
PubMed1999

Local striatal infusion of MPP+ does not result in increased hydroxylation after systemic administration of 4-hydroxybenzoate.

In vivo bilateral microdialysis in the rat striatum was used to investigate hydroxyl radical formation under basal conditions and after intrastriatal administration of the neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). After a short equilibration period, 4-hydroxybenzoate (4HBZ), which scavenges hydroxyl radicals to produce 3,4-dihydroxybenzoate (34DHB), was injected intraperitoneally 15 min before infusion of MPP+. To evaluate the enzymatic contribution to hydroxyl radical formation, two other series of microdialyses were performed following administration of monoamine oxidase B inhibitors, either 1-deprenyl (selegiline) or MDL 72,974A [(E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride]. Microdialysate samples were analyzed by high-performance liquid chromatography for catecholamines, 3,4-dihydroxyphenylacetate (DOPAC), homovanillate (HVA), along with the hydroxyl radical adduct, 34DHB and its precursor, 4HBZ. MPP+ administration resulted in a massive release of dopamine along with a decrease in DOPAC and HVA in all three groups. A striking effect in all three groups was noted in which MPP+ resulted in a decrease in interstitial 4HBZ to < 50% of the non-MPP+ -treated side. In absolute terms, the amount of 34DHB produced was low but similar in all three groups, even after unilateral MPP+ infusion. When 34DHB was normalized to 4HBZ release to account for differences in precursor availability, there were no significant differences in the 34DHB/4HBZ ratios either with or without MAO inhibitor treatment or after local MPP+ infusion. Systemic 4HBZ administration appears to result predominantly in intra-cellular sampling of hydroxyl radicals which produces different results from local infusion of trapping agents such as salicylate.

1-Methyl-4-phenylpyridinium↗
PubMed1994

Changes in the catalytic properties of p-hydroxybenzoate hydroxylase caused by the mutation Asn300Asp.

By site-directed mutagenesis, we have changed Asn300 to Asp in p-hydroxybenzoate hydroxylase (PHBH; EC 1.14.13.2) from Pseudomonas aeruginosa. In the wild-type (WT) enzyme, residue 300 is in contact with the isoalloxazine ring of the active-site FAD; in the Asn300Asp mutant, this side chain has moved by about 5 A, altering the protein structure [Lah, M.S., Palfey, B.A., Schreuder, H.A., & Ludwig, M.L. (1994) Biochemistry (following paper in this issue)]. The structural changes are responsible for profound catalytic and dynamic effects. The flavin of PHBH is reduced by NADPH in the first half of catalysis. The mutation has decreased this rate 330-fold, apparently by affecting the reactive orientation of the isoalloxazine and pyridine rings. Furthermore, the redox potential of the flavin is lower in the mutant enzyme than in WT by 20-40 mV. The reduced flavin of PHBH reacts with O2 to form a flavin C(4a)-hydroperoxide, which is the species that transfers oxygen to the aromatic substrate. Previous studies indicated that the enzyme promotes the hydroxylation reaction in part by activating the substrate through lowering the phenolic pKa. The Asn300Asp mutant does not lower the substrate pKa. As a consequence of this, and also an enhanced stability of the flavin C(4a)-hydroperoxide, the hydroxylation is 50-fold slower in the mutant than in WT. However, despite the slow rate of the hydroxylation reaction, no H2O2 is formed by the competitive elimination reaction. The kinetic stability of the flavin C(4a)-hydroxide formed by the hydroxylation was also enhanced by the mutation. By studying the effects of the inhibitor azide on the oxidative sequence, we were able to conclude that the inhibitory site is readily accessible to solvent; azide binding at a second site slowly displaces the substrate from the reduced enzyme. The mutation has profoundly slowed the rates of ligand binding to the enzyme. Kinetic studies of binding indicated the presence of several enzyme conformations. Thus, the mutation of this one residue interferes with the orientation of pyridine nucleotide and flavin during reduction, stabilizes flavin C(4a) intermediates, prevents substrate ionization, and alters the rates and strengths of ligand binding.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1994

Crystal structures of mutant Pseudomonas aeruginosa p-hydroxybenzoate hydroxylases: the Tyr201Phe, Tyr385Phe, and Asn300Asp variants.

Structures of the mutant p-hydroxybenzoate hydroxylases, Tyr201Phe, Tyr385Phe, and Asn300Asp, each complexed with the substrate p-OHB have been determined by X-ray crystallography. Crystals of these three mutants of the Pseudomonas aeruginosa enzyme, which differs from the wild-type Pseudomonas fluorescens enzyme at two surface positions (228 and 249), were isomorphous with crystals of the wild-type P. fluorescens enzyme, allowing the mutant structures to be determined by model building and refinement, starting from the coordinates for the oxidized P. fluorescens PHBH-3,4-diOHB complex [Schreuder, H.A., van der Laan, J.M., Hol, W.G.J., & Drenth, J. (1988) J. Mol. Biol. 199, 637-648]. The R factors for the structures described here are: Tyr385Phe, 0.178 for data from 40.0 to 2.1 A; Tyr201Phe, 0.203 for data from 40.0 to 2.3 A; and Asn300Asp, 0.193 for data from 40.0 to 2.3 A. The functional effects of the Tyr201Phe and Tyr385Phe mutations, described earlier [Entsch, B., Palfey, B.A., Ballou, D.P., & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349], were rationalized with the assumption that the mutations perturbed the hydrogen-bonding interactions of the tyrosine residues but caused no other changes in the enzyme structure. In agreement with these assumptions, the positions of the substrate, the flavin, and the modified residues are not altered in the Tyr385Phe and Tyr201Phe structures. In contrast, substitution of Asp for Asn at residue 300 has more profound effects on the enzyme structure. The side chain of Asp300 moves away from the flavin, disrupting the interactions of the carboxamide group with the flavin O(2) atom, and the alpha-helix H10 that begins at residue 297 is displaced, altering its dipole interactions with the flavin ring. The functional consequences of these changes in the enzyme structure and of the introduction of the carboxyl group at 300 are described and discussed in the accompanying paper (Palfey et al., 1994b).

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1982

A study of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: chemical modification of histidine residues.

The flavoprotein p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens is inactivated by diethyl pyrocarbonate. Below pH 7, diethyl pyrocarbonate reacts specifically with histidine residues. The inactivation reaction is biphasic and follows pseudo-first-order kinetics. Four of the nine histidine residues of the enzyme are modified. During the first phase of the reaction, one histidine residue is modified and leads to a loss of about 30% of the activity. Modification of the additional three histidine residues during the second phase leads to complete loss of activity. Two of the latter histidine residues are essential for activity and are involved in the binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH). The activity can be restored almost quantitatively upon treatment of modified enzyme with hydroxylamine. The modified enzyme is still capable of binding NADPH. The dissociation constant of the enzyme-NADPH complex is larger by a factor of 10 for the modified enzyme as compared to that for the native enzyme. The modification does not affect the affinity of the enzyme for the substrate, although effectors protect two histidine residues from chemical modification by diethyl pyrocarbonate. The rate of inactivation of the enzyme is pH dependent and increases with increasing pH values. From the pH dependence of the rate constant, it is calculated that two cooperative histidine residues participate in the reaction with diethyl pyrocarbonate. Both histidine residues possess a pKa' value of 6.2. At pH greater than 7, other reactions take place which are completely abolished in the presence of an effector (substrate) of the enzyme.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2002

DNA gyrase interaction with coumarin-based inhibitors: the role of the hydroxybenzoate isopentenyl moiety and the 5'-methyl group of the noviose.

DNA gyrase is a major bacterial protein that is involved in replication and transcription and catalyzes the negative supercoiling of bacterial circular DNA. DNA gyrase is a known target for antibacterial agents since its blocking induces bacterial death. Quinolones, coumarins, and cyclothialidines have been designed to inhibit gyrase. Significant improvements can still be envisioned for a better coumarin-gyrase interaction. In this work, we obtained the crystal costructures of the natural coumarin clorobiocin and a synthetic analogue with the 24 kDa gyrase fragment. We used isothermal titration microcalorimetry and differential scanning calorimetry to obtain the thermodynamic parameters representative of the molecular interactions occurring during the binding process between coumarins and the 24 kDa gyrase fragment. We provide the first experimental evidence that clorobiocin binds gyrase with a stronger affinity than novobiocin. We also demonstrate the crucial role of both the hydroxybenzoate isopentenyl moiety and the 5'-alkyl group on the noviose of the coumarins in the binding affinity for gyrase.

Anti-Infective Agents↗
PubMed1999

Use of free energy relationships to probe the individual steps of hydroxylation of p-hydroxybenzoate hydroxylase: studies with a series of 8-substituted flavins.

We report Hammett correlations, using 8-substituted flavins, to clarify the mechanism of hydroxylation by p-hydroxybenzoate hydroxylase (PHBH). The 8-position of the FAD isoalloxazine ring was chosen for modifications, because in PHBH it has minimal interactions with the protein, and it is accessible to solvent and away from the site of hydroxylation. Although two intermediates, a flavin-C4a-hydroperoxide and a flavin-C4a-hydroxide, are known to participate in hydroxylation, the mechanism of oxygen transfer remains controversial. Mechanisms as diverse as electrophilic aromatic substitution, diradical formation, and isoalloxazine ring opening have been proposed. In the studies reported here, it was possible to monitor spectrally each of the individual steps involved in hydroxylation, because the FAD cofactor acts as a reporter group. Thus, with PHBH, substituted separately with nine derivatives of FAD altered in the 8-position, quantitative structure-reactivity relationships (QSAR) have been applied to probe the mechanisms of formation of the flavin-C4a-hydroperoxide, the conversion to the flavin-C4a-hydroxide with concomitant oxygen transfer to the substrate, and the dehydration of the flavin-C4a-hydroxide to form oxidized FAD. The individual chemical steps in the mechanism of PHBH were not altered when using any of the modified flavins, and normal products were obtained; however, the rates of individual steps were affected, and depended on the electronic properties of the 8-substituent. Increased hydroxylation rates were observed when a more electrophilic flavin-C4a-hydroperoxide (i.e., with an electron-withdrawing substituent at the 8-position) is bound to PHBH. On the basis of QSAR analysis, we conclude that the mechanism of the hydroxylation step is best described by electrophilic aromatic substitution.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1999

Structure-function correlations of the reaction of reduced nicotinamide analogues with p-hydroxybenzoate hydroxylase substituted with a series of 8-substituted flavins.

Structural and kinetic studies have revealed two flavin conformations in p-hydroxybenzoate hydroxylase (PHBH), the in-position and the out-position. Conversion between these two conformations is believed to be essential during catalysis. Although substrate hydroxylation occurs while the flavin in PHBH is in the in-conformation, the position of the flavin during reduction by NADPH is uncertain. To investigate the catalytic importance of the out-conformation of the flavin and to clarify the mechanism of flavin reduction in PHBH, we report quantitative structure-reactivity relationships (QSAR) using PHBH substituted separately with nine derivatives of FAD modified in the 8-position and four dihydronicotinamide analogues as reducing agents. The 8-position of the FAD isoalloxazine ring was chosen for modification because in PHBH it has minimal interactions with the protein and is accessible to solvent. The chemical sequence of events during catalysis by PHBH was not altered when using any of the modified flavins, and normal products were obtained. Although the rate of reduction of PHBH reconstituted with flavin derivatives is expected to be dependent on the redox potential of the flavin, no strict correlation was observed. Instead, the rate of reduction correlated with the kappa-substituent constant, which is based on size and hydrophobicity of the 8-substituent on the FAD. Substituents that sterically hinder attainment of the out-conformation decreased the rate of flavin reduction much more than expected on the basis of the redox potential of the flavin. The results of this QSAR analysis are consistent with the hypothesis that the flavin in PHBH must move to the out-conformation for proper formation of the charge-transfer complex between NADPH and FAD that is necessary for rapid flavin reduction.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2002

Characterization of the early stage aminoshikimate pathway in the formation of 3-amino-5-hydroxybenzoic acid: the RifN protein specifically converts kanosamine into kanosamine 6-phosphate.

The biosynthesis of 3-amino-5-hydroxybenzoic acid (AHBA), precursor of the ansamycin and mitomycin antibiotics, proceeds by the aminoshikimate pathway from 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP). Identification of RifN, product of one of three genes from the rifamycin biosynthetic gene cluster known to be essential for aminoDAHP formation, as a specific kanosamine (3-deoxy-3-amino-D-glucose) 6-kinase establishes kanosamine and its 6-phosphate as specific intermediates in AHBA formation. This suggests a hypothetical reaction sequence for aminoDAHP formation, and thus for the early steps of AHBA biosynthesis, starting from UDP-D-glucose and introducing the nitrogen by oxidation and transamination at C-3.

Actinomycetales↗
PubMed2005

Conformational dynamics of the isoalloxazine in substrate-free p-hydroxybenzoate hydroxylase: single-molecule studies.

p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric enzyme in which each subunit noncovalently binds one molecule of FAD in the active site. PHBH is a model system for how flavoenzymes regulate reactions with oxygen. We report single-molecule fluorescence studies of PHBH in the absence of substrate that provide data consistent with the hypothesis that a critical step in substrate binding is the movement of the isoalloxazine between an "in" conformation and a more exposed or "open" conformation. The isoalloxazine is observed to move between these conformations in the absence of substrate. Studies with the Y222A mutant form of PHBH suggest that the exposed conformation is fluorescent while the in-conformation is quenched. Finally, we note that many of the single-molecule-fluorescence trajectories reveal a conformational heterogeneity, with populations of the enzyme characterized by either fast or slow switching between the in- and open-conformations. Our data also allow us to hypothesize a model in which one flavin in the dimer inhibits the motion of the other.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2003

Three novel hydroxybenzoate saxitoxin analogues isolated from the dinoflagellate Gymnodinium catenatum.

In a recent survey of paralytic shellfish poisoning (PSP) toxins in Gymnodinium catenatum Graham extracts, using LC with postcolumn oxidation and fluorescence detection, three novel saxitoxin analogues were revealed in isolates from several locations, including Australian waters. We have named them as G. catenatum toxins, GC1 (1), GC2 (2), and GC3 (3). The compounds were isolated from a culture of the Australian strain by LC-MS-guided fractionation employing a C18-silica column and hydrophilic interaction chromatography. The unusual structures of these novel compounds were characterized by low- and high-resolution MS, MS/MS, and NMR spectroscopy. GC3 (3) was found to be the 4-hydroxybenzoate ester derivative of decarbamoylsaxitoxin, while GC1 (1) and GC2 (2) are the epimeric 11-hydroxysulfate derivatives of GC3 (3).

Animals↗