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PubMed1967

The relationship of 4-hydroxybenzoic acid to lysine and methionine formation in Escherichia coli.

1. A multiple aromatic mutant, Escherichia coli 156:53D2, required 4-hydroxybenzoic acid for rapid aerobic growth on a number of carbon sources. 2. In the absence of 4-hydroxybenzoic acid aerobic growth was stimulated by a mixture of lysine and methionine and by succinate. The influence of the amino acids is attributed to a sparing of succinyl-CoA. 3. Low activities of both alpha-oxoglutarate dehydrogenase and fumarate reductase were found in organisms grown aerobically without 4-hydroxybenzoate and consequently both mechanisms known for the formation of succinate were impaired. 4. The low fumarate-reductase activity in these organisms was due to repression of enzyme synthesis by aeration and not to enzyme inactivation. In contrast lactate dehydrogenase and ethanol dehydrogenase were induced. This is interpreted as the appearance of alternative routes of NADH oxidation when electron transfer to oxygen is impaired. 5. The activities of the other tricarboxylic acid-cycle enzymes tested were little influenced by 4-hydroxybenzoate deficiency, although anaerobiosis resulted in a fall in activity.

Alcohol Oxidoreductases↗
PubMed2001

The activity of Arabidopsis glycosyltransferases toward salicylic acid, 4-hydroxybenzoic acid, and other benzoates.

Benzoates are a class of natural products containing compounds of industrial and strategic importance. In plants, the compounds exist in free form and as conjugates to a wide range of other metabolites such as glucose, which can be attached to the carboxyl group or to specific hydroxyl groups on the benzene ring. These glucosylation reactions have been studied for many years, but to date only one gene encoding a benzoate glucosyltransferase has been cloned. A phylogenetic analysis of sequences in the Arabidopsis genome revealed a large multigene family of putative glycosyltransferases containing a consensus sequence typically found in enzymes transferring glucose to small molecular weight compounds such as secondary metabolites. Ninety of these sequences have now been expressed as recombinant proteins in Escherichia coli, and their in vitro catalytic activities toward benzoates have been analyzed. The data show that only 14 proteins display activity toward 2-hydroxybenzoic acid, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid. Of these, only two enzymes are active toward 2-hydroxybenzoic acid, suggesting they are the Arabidopsis salicylic acid glucosyltransferases. All of the enzymes forming glucose esters with the metabolites were located in Group L of the phylogenetic tree, whereas those forming O-glucosides were dispersed among five different groups. Catalytic activities were observed toward glucosylation of the 2-, 3-, or 4-hydroxyl group on the ring. To further explore their regioselectivity, the 14 enzymes were analyzed against benzoic acid, 3-hydroxybenzoic acid, 2,3-, 2,4-, 2,5-, and 2,6-dihydroxybenzoic acid. The data showed that glycosylation of specific sites could be positively or negatively influenced by the presence of additional hydroxyl groups on the ring. This study provides new tools for biotransformation reactions in vitro and a basis for engineering benzoate metabolism in plants.

Arabidopsis↗
PubMed1980

On the effect of cellular nucleophiles on the binding of metabolites of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene and 9-hydroxybenzo(a)pyrene to nuclear DNA.

The binding to DNA of products resulting from the further activation of trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene and 9-hydroxybenzo(a)pyrene was studied in several incubation systems. In a system containing purified DNA and rat liver microsomes, products of 9-hydroxybenzo(a)pyrene were the predominant binding species. In a system containing isolated rat hepatocytes, the total binding was much lower, and products of trans-7,8-dihydroxy-7, 8-dihydrobenzo(a)pyrene predominated. Both the total amounts and the ratios of the bound species were altered by the addition of various soluble nucleophiles to the incubation system. The binding of 9-hydroxybenzo(a)pyrene to both nuclear and purified DNA was decreased in the presence of "non-specific" protein in the incubate. A decrease in the binding of trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene to either purified or nuclear DNA was seen after the addition of active cytosol, but not with protein alone. Either denaturation of the cytosol, or depletion of glutathione by diethylmaleate treatment, partially negated this effect. We conclude that the binding of benzo(a)pyrene metabolites to DNA in the cell is decreased by soluble nucleophiles, and that this trapping of metabolites is selective. 9-Hydroxybenzo(a)pyrene metabolites are removed by non-specific protein binding, whereas removal of trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene metabolites requires higher affinity binding or enzymatic conjugation.

Animals↗
PubMed1994

Biosynthesis of p-Hydroxybenzoate from p-Coumarate and p-Coumaroyl-Coenzyme A in Cell-Free Extracts of Lithospermum erythrorhizon Cell Cultures.

The enzymatic formation of p-hydroxybenzoate from p-coumarate in cell-free extracts of cell cultures of Lithospermum erythrorhizon Sieb. et Zucc. was investigated. p-Coumaroyl-coenzyme A (p-coumaroyl-CoA) is the activated intermediate in this biosynthetic reaction. It is formed by an ATP-, Mg2+ -, and CoA-dependent 4-hydroxycinnamate:CoA ligase reaction. p-Coumaroyl-CoA is oxidized and cleaved to p-hydroxybenzoyl-CoA and acetyl-CoA in a thioclastic reaction in which NAD is an essential cofactor. These CoA esters are rapidly hydrolyzed to acetate and p-hydroxybenzoate, probably by thioesterases. The enzymes involved in the formation of p-hydroxybenzoate are soluble. p-Hydroxybenzalde-hyde is not an intermediate in this conversion, and S-denosylmethionine and uridine-5[prime]-diphosphoglucose do not enhance formation of p-hydroxybenzoate in our system.

Journal Article↗
PubMed2000

meso-5,5,7,12,12,14-Hexamethyl-1,4,8,11-tetraazacyclotetradecane as a building block in supramolecular chemistry; salts formed with 2,2-biphenol, 4,4-thiodiphenol, 4,4-sulfonyldiphenol, 3-and 4-hydroxybenzoic acids, 3,5-dihydroxybenzoic acid and phenylphosphonic acid; supramolecular structures in zero, one, two and three dimensions

The structure of meso-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane-2,2'-biphenol (1/2), (C16H36N4).(C12H10O2)2 (1), is a salt [C16H38N4]2+.2[HOC6H4C6H4O]-: the cations are centrosymmetric with two protons held within the N4 cavity of the macrocycle by N-H...N hydrogen bonds, and the phenolate anions contain intramolecular O-H...O-hydrogen bonds. The ions are linked into a finite centrosymmetric aggregate by means of N-H...O hydrogen bonds. meso-5,5,7,12,12,14-Hexamethyl-1,4,8,11-tetraazacyclotetradecane-4,4'-thiobiphenol-methanol (1/2/2), (C16H36N4).(C12H10O2S)2.(CH4O)2 (2), and meso-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane-4,4'-sulfonylbiphenol-methanol (1/2/2), (C16H36N4).(C12H10O4S)2.(CH4O)2 (3), are isomorphous: each is a salt, [C16H38N4]2+.2[HOC6H4SC6H4O]-.2MeOH (2) and [C16H38N4]2+.2[HOC6H4SO2C6H4O]-.2MeOH (3), and in each the phenolate anions are linked by O-H...O- hydrogen bonds into chains; antiparallel pairs of chains are cross-linked by the cations to form molecular ladders, with neutral methanol molecules acting as spacer units. In meso-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane-3-hydroxybenzoic acid-methanol (1/2/2) (4), 3-hydroxybenzoate anions form chains, again cross-linked in pairs by the [C16H38N4]2+ cations to form molecular ladders, different from those in (2) and (3) in that the neutral methanol units are pendent from the ladders, rather than forming a part of it. meso-5,5,7,12,12,14-Hexamethyl-1,4,8,11-tetraazacyclotetradecane-4-hydroxybenzoic acid-methanol (1/2/1) (5) is again a salt, [C16H38N4]2+.2[HOC6H4COO]-.MeOH: chains of 4-hydroxybenzoate anions are continuously cross-linked by two different types of [C16H38N4]2+ cation into a two-dimensional net. Only one of the two types of cation is linked to the chains via neutral methanol spacer units. meso-5,5,7,12,12,14-Hexamethyl-1,4,8,11-tetraazacyclotetradecane-phenylphosphonic acid-water (1/4/2) (6) is a salt, [C16H40N4]4+.4[C6H5PO3H]-.2H2O, containing the centrosymmetric tetraprotonated amine units, which have a conformation quite different from the trans-III conformation uniformly found in the [C16H38N4]2+ cations. The phenylphosphonate anions and the water molecules are linked into chains of fused rings, which are linked by the cations into two-dimensional nets. In meso-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetrade cane-3,5-dihydroxybenzoic acid (1/2) (7), the 3,5-dihydroxybenzoate anions in the unsolvated salt [C16H38N4]2+.2[(HO)2C6H3COO]- are linked into continuous two-dimensional nets, which are in turn linked by the centrosymmetric cations to form a three-dimensional framework. meso-5,5,7,12,12,14-Hexamethyl-1,4,8,11-tetraazacyclo tetradecane-4,4'-biphenol (1/3) (8) is a salt containing both neutral and anionic biphenol units, [C16H38N4]2+.2[HOC6H4-C6H4O]-.[HOC6H4C6H4OH]. The two types of biphenol unit form two-dimensional nets and these nets are linked by the cations to form three independent, three-dimensional frameworks which are fully interwoven, but not bonded to one another.

Journal Article↗
PubMed1994

Reversible Conversion of 4-Hydroxybenzoate and Phenol by Clostridium hydroxybenzoicum.

Reversible conversion of 4-hydroxybenzoate and phenol and their analogs was observed in whole-cell suspensions and cell extracts of Clostridium hydroxybenzoicum grown with 4-hydroxybenzoate and 3,4-dihydroxybenzoate. Assuming that bicarbonate is the cosubstrate, the equilibrium constants calculated for the reactions 4-hydroxybenzoate + H(2)O left arrow over right arrow phenol + HCO(3) and 3,4-dihydroxybenzoate + H(2)O left arrow over right arrow catechol + HCO(3) were 11.4 (+/- 0.5) and 5.05 (+/- 0.25), respectively. In a phenol-adapted sediment slurry, 4-hydroxybenzoate and 3,4-dihydroxybenzoate were decarboxylated to phenol and to catechol, respectively, as intermediates without a lag time.

Journal Article↗
PubMed1988

Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The oxidation-reduction potential of p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and Müller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1991

Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants.

The role of protein residues in activating the substrate in the reaction catalyzed by the flavoprotein p-hydroxybenzoate hydroxylase was studied. X-ray crystallography (Schreuder, H. A., Prick, P.A.J., Wieringa, R.K., Vriend, G., Wilson, K.S., Hol, W.G. J., and Drenth, J. (1989) J. Mol. Biol. 208, 679-696) indicates that Tyr-201 and Tyr-385 form a hydrogen bond network with the 4-OH of p-hydroxybenzoate. Therefore, site directed mutants were constructed, converting each of these tyrosines into phenylalanines. Spectral (visible and fluorescence) properties, reduction potentials, and binding constants are very similar to those of wild type, indicating that there are no major structural changes in the mutants. In the absence of substrate, the mutants and wild type exhibit similar pH-dependent changes in the FAD spectrum. However, the enzyme-substrate complex of Tyr-201----Phe lacks an ionization observed in both wild type and Tyr-385----Phe, which preferentially bind the phenolate form of substrates. Tyr-201----Phe shows no preference, indicating that Tyr-201 is required to ionize the substrate. The mutants have less than 6% the activity of the wild type enzyme. The effects on catalysis were studied by stopped flow techniques. Reduction of FAD by NADPH is slower by 10-fold in Tyr-201----Phe and 100-fold in Tyr-385----Phe. When the reduced Tyr-201----Phe-p-hydroxybenzoate complex reacts with oxygen, a long-lived flavin-C(4a)-hydroperoxide is observed, which slowly eliminates H2O2 with very little hydroxylation. Thus, the role of Tyr-201 is to activate the substrate by stabilizing the phenolate. Tyr-385----Phe reacts with oxygen to form 25% oxidized enzyme, and 75% flavin hydroperoxide, which successfully hydroxylates the substrate. This mutant also hydroxylates the product (3, 4-dihydroxybenzoate) to form gallic acid.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1976

Catalytic mechanism of p-hydroxybenzoate hydroxylase with p-mercaptobenzoate as substrate.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens catalyzes in vivo the hydroxylation of p-hydroxybenzoate by molecular oxygen to form 3,4-dihydroxybenzoate. p-Mercaptobenzoate is also a substrate of the enzyme, but instead of being converted to the expected product, 3-hydroxy-4-mercaptobenzoate, the disulfide, 4,4'-dithiobisbenzoate, is formed. To find what mechanistic information this unusual reaction provided, steady state kinetic analyses, combined with rapid reaction studies of the changes in the enzyme-bound FAD, were carried out with the separate half-reactions involved in catalysis. Most of the kinetic measurements were made with a stopped-flow spectrophotometer designed for working anaerobically and connected on line to a minicomputer. Initial rate studies, upon varying systematically the concentrations of p-mercaptobenzoate, NADPH, and oxygen showed that the enzyme interacted with the substrates in the same manner as it does with p-hydroxybenzoate in place of the mercaptan. That is, a ternary complex is formed between enzyme, mercaptobenzoate, and NDAPH, followed by reaction and release of NADP+. Then a second ternary complex is formed between enzyme, mercaptobenzoate, and oxygen followed by reaction, liberation of product, and return to the resting state of the enzyme. Rapid reaction studies showed that the first half-reaction was analagous to that with the natural substrate. The enzyme-flavin is reduced to the 1,5-dihydroflavin by NADPH, and the rate of reaction is dramatically enhanced in the presence of mercaptobenzoate. The rate enhancement with this enzyme correlates well with the presence of a dianion form of the substrate on the enzyme. Examination of the second half-reaction showed that the reduced flavin on the enzyme formed transient intermediates upon reaction with oxygen, which were analogous to the intermediates in reactions where the enzyme forms an hydroxylated product. The oxidation of p-mercaptobenzoate by H2O2 in free solution resulted in the same disulfide as formed in the enzymatic reaction, only orders of magnitude slower. A sulfenic acid was probably the initial oxidation product from p-mercaptobenzoate, and this reacted very fast, and nonenzymatically, with mercaptobenzoate to form the disulfide and H20. The significance of the enzyme reaction with oxygen when complexed with p-mercaptobenzoate is discussed in relation to the mechanism of hydroxylation.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMedDate not supplied

Molecular imprinting of nitrophenol and hydroxybenzoic acid isomers: effect of molecular structure and acidity on imprinting.

Three nitrophenol isomer-imprinted polymers were prepared under the same conditions using 4-vinylpyridine as a functional monomer. Different recognition capacities for template molecules were observed for the three polymers. Another imprinting system with stronger acidity than nitrophenol isomers, 2-hydroxybenzoic acid (salicylic acid) and 4-hydroxybenzoic acid, was imprinted using 4-vinylpyridine or acrylamide as functional monomer respectively. Both 4-hydroxybenzoic acid-imprinted polymers using the two monomers showed recognition ability for the template molecule. However, when acrylamide was chosen as functional monomer, the salicylic acid-imprinted polymer showed very weak recognition for the template molecule, whereas strong recognition ability of the resultant polymer for salicylic acid was observed with 4-vinylpyridine as functional monomer. It seems that the structure and acidity of template molecules is responsible for the difference in recognition, by influencing the formation and strength of interaction between template molecule and functional monomer during the imprinting process. An understanding of the mechanism of molecular imprinting and molecular recognition of MIPs will help to predict the selectivity of MIPs on the basis of template molecule properties.

Chromatography, High Pressure Liquid↗
PubMed1983

Extended Hildebrand solubility approach: p-hydroxybenzoic acid in mixtures of dioxane and water.

The extended Hildebrand solubility approach was used to reproduce the solubilities of p-hydroxybenzoic acid in a dioxane-water system. The solubility parameter of p-hydroxybenzoic acid was determined and found to be approximately 15 (cal/cm3)1/2. Residual plots (scattergrams) were used in conjunction with R2, F, and standard deviation values to determine whether a quadratic, cubic, quartic, or higher degree polynomial was required in the calculations. The earlier iteration method for back-calculations of solubilities was replaced by the more reliable root-finder method. The solubility profile of p-hydroxybenzoic acid in dioxane-water mixtures did not follow a log linear relationship even in the ranges where the solubility parameters of the water-cosolvent mixture might be expected to produce a straight-line function, as observed in other studies.

Chemical Phenomena↗
PubMed1992

Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase refined at 2.3 A resolution.

The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme-substrate complex in deaerated mother liquor containing 300-400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme-bound FAD by NADPH. This was confirmed by single crystal spectroscopy. X-ray data to 2.3 A were collected on oscillation films using a rotating anode generator as an X-ray source. After data processing and reduction, restrained least squares refinement using the 1.9 A structure of the oxidized enzyme-substrate complex as a starting model, yielded a crystallographic R-factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p-hydroxybenzoate and 322 solvent molecules. The structures of the oxidized and reduced forms of the enzyme-substrate complex were found to be very similar. The root-mean-square discrepancy for all atoms between both structures was 0.38 A. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2 degrees. This value should be compared with observed values of 10 degrees for the oxidized enzyme-substrate complex and 19 degrees for the enzyme-product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1990

Effect of fluorinated analogues of phenol and hydroxybenzoates on the anaerobic transformation of phenol to benzoate.

The effects of fluorinated analogues on the anaerobic transformation of phenol to benzoate were examined. At greater than or equal to 250 microM 2- or 3-fluorophenol, phenol transformation was delayed. 2-Fluorophenol had no apparent effect on subsequent degradation of benzoate, but benzoate accumulated in the presence of greater than or equal to 250 microM 3-fluorophenol. In contrast, 4-fluorophenol at less than or equal to 2 mM had no effect on either phenol transformation or benzoate degradation. Phenol and 2-, or 3-fluorophenol were transformed simultaneously, but phenol was transformed more rapidly than either fluorophenol. Thus, fluorinated analogues of phenol did not prevent anaerobic transformation of phenol to benzoate. 2-Fluorophenol was converted to 3-fluorobenzoate, and phenol enhanced the rate and extent of its transformation. 3-Fluorophenol was transformed to 2-fluorobenzoate to a limited extent (approximately 3%) when phenol was present. 4-Fluorophenol was not transformed regardless of the presence of phenol. 3-Fluoro-4-hydroxybenzoate, a potential fluorinated intermediate product of para-carboxylation, was transformed rapidly to 2-fluorophenol and 3-fluorobenzoate, irrespective of the presence of phenol, indicating that both dehydroxylation and decarboxylation occurred. Initially, 2-fluorophenol and 3-fluorobenzoate were rapidly formed in an approximate molar ratio of 2:1. Once 3-fluoro-4-hydroxybenzoate was completely removed, the 2-fluorophenol, initially formed, was converted to 3-fluorobenzoate at a slower rate. Thus, phenol enhanced transformation of the fluorinated analogues, and the products of transformation suggested para-carboxylation. 3-Fluoro-2-hydroxybenzoate was not transformed in either the presence or absence of phenol, indicating that ortho-carboxylation did not occur.

Anaerobiosis↗
PubMed1992

Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeasts Candida parapsilosis and Arxula adeninivorans: evidence for an operative gentisate pathway.

The imperfect ascomycetous yeasts Candida parapsilosis and Arxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxybenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid by C. parapsilosis.

Biodegradation, Environmental↗
PubMed1975

[On the phenolic acids of vegetables. IV. Hydroxycinnamic acids and hydroxybenzoic acids of vegetables and potatoes (author's transl)].

Lettuce, endive and chicory exclusively, cornsalad and sweet fennel almost exclusively contain caffeic acid derivatives beside traces of ferulic acid. Parsley exclusively and spinach almost exclusively show p-coumaric acid derivatives. Compared to root, fruit and seed vegetables the contents of phenolic acids in green leaves are considerably high. Rhubarb is the only vegetable, which contains gallic acid (chief phenolic acid) beside hydroxycinnamic, protocatechuic and vanillic acid derivatives. Furthermore hydroxybenzoic acid derivatives (salicylic, gentisic and vanillic acid) occur in cornsalad, sweet fennel, parsley and spinach in small concentrations; cornsalad shows p-hydroxybenzoic acid (ca. 20 mg/kg). Onions (Allium cepa) contain almost only protocatechuic acid beside small amounts of p-hydroxybenzoic and vanillic acid. In the outer dry coloured skins protocatechuic acid reaches concentrations up to 2% of plant material; the internal pulpy tissues show lower concentrations (ca. 20 mg/kg). On the contrary to the bulbs the green leaves of onions like chive and leek contain almost exclusively compounds of ferulic and p-coumaric acid. Garlic even shows a different phenolic acid pattern of skins and internal tissues. The caffeic acid derivatives of potatoes are mainly localized to a 1--2 mm thick outer layer. The different localization of phenolic acids in the different parts of vegetable plants is discussed.

Caffeic Acids↗
PubMed1975

[On phenolic acids of vegetables. I. Hydroxycinnamic acids and hydroxybenzoic acids of brassica-species and leaves of other cruciferae (author's transl)].

The contents of phenolic acids in vegetables of the species Brassica almost totally consist of hydroxycinnamic acid compounds. In contrary to other species of vegetables sinapic acid is dominant. Leaves of radish (Rhaphanus sativus var. sativus and var. niger) mainly contain compounds of caffeic and p-coumaric acid; leaves of horse radish show only traces of hydroxycinnamic acids. In the group of hydroxybenzoic acid derivatives traces of salicylic and gentistic acid could be determined in almost all species and frequently vanillic acid. Protocatechnic acid was only identified in red cabbage, especially in the head, syringic acid in gardencress and p-hydroxybenzoic acid in horse radish leaves. No other hydroxybenzoic acids or hydroxycoumarins could be detected.

Caffeic Acids↗
PubMed1995

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from a Klebsiella pneumoniae mutant strain.

Unlike the parent wild-type strain, the Klebsiella pneumoniae mutant strain MAO4 has a 4-HBA+ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when the Acinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoate K. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1989

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes.

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Hydroxybenzoate-3-Monooxygenase↗