P-hydroxybenzoate Hydroxylase Research Articles

An essential arginine residue at the substrate-binding site of p-hydroxybenzoate hydroxylase.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) was rapidly inactivated by treatment with phenylglyoxal, by a process obeying pseudo-first order kinetics. The reaction with the reagent was also examined by amino acid analyses, radioactivity measurements, and spectrophotometric analyses. Results of these analyses were consistent with each other, which shows that the inactivation was due to modification of argiine residue(s). Addition of saturating amounts of p-hydroxybenzoate (or benzoate) during the treatment resulted in marked protection of the enzyme from the inactivation as well as a significant decrease in modification of arginine residues, while phenol showed no effect. Modification in the absence of p-hydroxybenzoate caused a spectral change in the flavin moiety of the enzyme similar to that due to the enzyme.substrate complex formation, and losses in both the overall activity and the substrate-binding ability accompanied the spectral change. On the other hand, such spectral change was not observed and the substrate-binding ability was retained even after the overall activity had decreased to a great extent when p-hydroxybenzoate as added during the modification treatment. These results suggest that phenylglyoxal (an analogue of the substrate) was incorporatd into the substrate-binding site and that an arginine residue is involved in the site, having an interaction with the carboxylate anion of the substrate.

Fluoride elimination from substrates in hydroxylation reactions catalyzed by p-hydroxybenzoate hydroxylase.

Several fluorinated derivatives of p-hydroxybenzoate were synthesized and examined as substrates in the reaction catalyzed by p-hydroxybenzoate hydroxylase. All the derivatives tested served as substrates, undergoing tightly coupled hydroxylation by molecular oxygen. Hydroxylation of the difluoro and tetrafluoro derivatives liberated stoichiometric amounts of fluoride. Little or no fluoride was released with monofluoro substrates. The defluorination caused higher consumption of NADPH with an overall NADPH to oxygen ratio of 2, in contrast to the ratio of 1 with the physiological substrate and with the monofluoro derivatives. Evidence was obtained strongly suggestive of a quinonoid species as the primary product formed upon oxygenative defluorination. The additional equivalent of NADPH consumed upon fluoride elimination is presumably used in a nonenzymatic reaction with the quinonoid intermediate, resulting in the observed dihydroxy product. Stopped flow studies of the reductive and oxidative half-reactions with tetrafluoro-p hydroxybenzoate substrate were examined. The oxygen half-reaction was analogous to that with p-hydroxybenzoate involving two transient oxygenated flavin intermediates. The decay of the first intermediate, a C(4a)-peroxyflavin, results in rupture of the oxygen-oxygen bond and is rate-determining in overall catalysis. This is in contrast to the reaction with the normal substrate, presumably due to a deactivating effect of the fluorine substituents. The above results are consistent with an oxenoid mechanism of oxygen attack.

Photodehalogenation of 7- and 8-halogen-substituted flavins. Photochemistry of the reduced flavin chromophore.

Flavodoxin was reconstituted with 8-chloro- and 7-bromo-FMN and p-hydroxybenzoate hydroxylase with the analogous FAD derivatives. In all cases, the spectral properties of the artificial enzymes changed as a result of photoreduction in the presence of ethylenediaminetetraacetate or oxalate as sources of reducing equivalents. The same changes were found to occur on irradiation of the enzymes which had been reduced previously in the dark under anaerobic conditions with dithionite. Using analogous 7- and 8-chlorolumiflavins, the observed changes were shown to be due to a novel photoreaction of the reduced flavin chromophore, in which either the 7- or 8-halogen substituent is eliminated and replaced by a proton derived from the solvent. The same reaction was shown to occur with 7,8-bis-norlumiflavin where 1 deuterium atom was incorporated into the molecule as a result of photoirradiation of the reduced flavin in deuterated medium. In the case of p-hydroxybenzoate hydroxylase, both the 8-chloro-FAD and 7-bromo-FAD enzymes, as well as their 8-nor-FAD and 7-nor-FAD photoproducts, possessed catalytic activity comparable to that of the native enzyme.

Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD.

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.

A study of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Improved purification, relative molecular mass, and amino acid composition.

The purification procedure for p-hydroxybenzoate hydroxylase has been modified by replacement of the DEAE-cellulose (DE-32) column in the original procedure by a Sephadex--Cibacron-blue affinity column. In this way the yield of enzyme could be improved from 16% to about 40--50%. Preparative gel chromatography indicated that the enzyme does not exist as a monomeric species as earlier believed but mainly as a dimer. Sodium dodecyl sulfate gel electrophoresis of purified enzyme revealed a minimum relative molecular mass (Mr) of 43000--45000. Analytical gel chromatography, sedimentation equilibrium and sedimentation velocity experiments showed that the enzyme exists in solution mainly as a dimer but also in higher-order quaternary structures (presumably tetramer and hexamer). Temperature dependence of the distribution of the oligomers suggests that the association is of hydrophobic nature. The amino acid composition of the enzyme is also presented. The enzyme contains no disulfide but five sulfhydryl groups. In the native state of the enzyme only one sulfhydryl group is accessible to N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid). The iso-electric point of the enzyme was found to be 5.8.

Studies on the interaction of p-hydroxybenzoate hydroxylase with NADPH. Effects of pH and substrates on the enzyme . NADPH complex formation.

Studies on the interaction of p-hydroxybenzoate hydroxylase with NADPH. Effects of pH and substrates on the enzyme . NADPH complex formation.

Kinetic studies on the reaction of p-hydroxybenzoate hydroxylase. Agreement of steady state and rapid reaction data.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens is a NADPH-dependent, FAD-containing monooxygenase catalyzing the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate in the presence of NADPH and molecular oxygen. The mechanism of this three-substrate reaction was investigated in detail at pH 6.6, 4 degrees C, by steady state kinetics, stopped flow spectrophotometry, and equilibrium binding experiments. The initial velocity patterns are consistent with a ping-pong type mechanism which involves two ternary complexes between the enzyme and substrates. The first ternary complex is formed by random addition of p-hydroxybenzoate and NADPH to the enzyme, followed by the release of the first product (NADP+). The reduced enzyme . p-hydroxybenzoate complex now reacts with oxygen, the third substrate, to form the second ternary complex. The enzyme-bound p-hydroxybenzoate then reacts with the activated oxygen to give 3,4-dihydroxybenzoate which is released regenerating the oxidized enzyme for the next cycle. The binding of p-hydroxybenzoate to the oxidized enzyme to form a 1:1 complex causes large, characteristic spectral perturbations and fluorescence quenching. The dissociation constant for the enzyme . substrate complex was obtained by titrations in which absorbance and/or fluorescence quenching was measured. The binding constants of NADPH to the enzyme with and without p-hydroxybenzoate were determined kinetically by measuring the rate of reduction of the enzyme at different concentrations of NADPH. The reduction of the enzyme proceeds extremely slowly in the absence of p-hydroxybenzoate. The presence of the substrate causes a dramatic stimulation (140,000-fold) in the rate of enzyme reduction. The anaerobic reduction of the enzyme by NADPH in the presence of p-hydroxybenzoate produces a transient charge-transfer intermediate. On the basis of the proposed mechanism, the dissociation constants for p-hydroxybenzoate and NADPH as well as the Michaelis constants for all the three substrates were calculated from the initial velocity data. The agreement obtained between various kinetic parameters from the initial rate measurements and those calculated from the individual rate constants determined in rapid reactions, strongly supports the proposed mechanism for the p-hydroxybenzoate hydroxylase reaction.

Crystal structure of p-hydroxybenzoate hydroxylase

The structure of the enzyme p-hydroxybenzoate hydroxylase (EC 1.14.13.2) in a complex with its substrate has been determined at a resolution of 2.5 Å. The molecular weight is 43,000 and the dimensions of one molecule are approximately 70 Å × 50 Å × 45 Å. The crystal structure contains dimers of these molecules. Approximately 16% of the residues occur in β-sheets and 26% in α-heliees. The molecule can be divided into three domains. The active site, near the isoalloxazine ring, is formed by side-chains of the three domains. The N-5 edge of the isoalloxazine ring points to p-hydroxybenzoate, which is bound in a deep cleft.

On the stable enzyme-substrate complex of p-hydroxybenzoate hydroxylase. Evidences for the proton uptake from the substrate.

On the stable enzyme-substrate complex of p-hydroxybenzoate hydroxylase. Evidences for the proton uptake from the substrate.

53] p-Hydroxybenzoate hydroxylase and melilotate hydroxylase

Publisher Summary This chapter focuses on the p-hydroxybenzoate hydroxylase and melilotate hydroxylase. Crystalline preparations of p-hydroxybenzoate hydroxylase have been obtained from four different species of Pseudomonas : P. desmolytica, P. putida A 3.12, putida M-6, and P. fluorescens. The enzymes from both P. putida species are extremely unstable undergoing rapid inactivation unless protected by substrates and other stabilizing agents, thereby making any detailed mechanistic investigation difficult. In contrast, the P. florescens enzyme described in the chapter is stable for months even in the absence of stabilizing agents. Several of these hydroxylases have been purified and characterized. Those obtained in pure stable form and studied include salicylate hydroxylase, p-hydroxybenzoate hydroxylase, and melilotate hydroxylase. A number of microorganisms capable of utilizing phenolic molecules as a sole source of carbon contain flavoprotein hydroxylases. The activity of the enzyme is determined spectrophotometrically by following the rate of substrate-dependent oxidation of NADPH at 340 nm. The enzyme activity can also be assayed by measuring the rate of oxygen consumption using an oxygen electrode.

On the structure of flavin-oxygen intermediates involved in enzymatic reactions.

During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421--505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C(4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of D-amino acid oxidase, p-hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the subsituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavoproteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides.

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.

Flavin-oxygen derivatives involved in hydroxylation by p-hydroxybenzoate hydroxylase.

Para-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens is one of a group of flavoproteins which insert molecular oxygen into aromatic rings to form phenols. To determine the mechanism of oxygen insertion by this enzyme, an extensive study was made of the reaction with O2 of reduced enzyme in complex with various aromatic molecules. Reactions were studied by following absorbance changes with time with a stopped-flow spectrophotometer. Analysis of multiphasic reactions led to the detection of a minimum of three transient intermediates with characteristic absorption spectra involved in the process of hydroxylation. The initial interaction of oxygen with the reduced enzyme characteristically produces a derivative of FAD (maximum absorbance 380 to 390 nm) which is probably C(4a) peroxyflavin. Depending on the aromatic compound bound to the enzyme, this intermediate decays either to oxidized, enzyme-bound flavin and H2O2 or transfers an atom of oxygen to the aromatic compound. The process of oxygen transfer forms a derivative of FAD of unknown structure (maximum absorbance 390 to 420 nm), which subsequently decays to the third intermediate observed (maximum absorbance 380 to 385 nm), which is probably C(4a) hydroxyflavin. The decay of this last intermediate results in the formation of oxidized enzyme, and the liberation of hydroxylated product and H2O. In an extension of substrate specificity studies it was found that p-aminobenzoate is a substrate and 5-hydroxypicolinate is an effector for p-hydroxybenzoate hydroxylase. The binding of aromatic compounds to the reduced enzyme was observed by following shifts in the absorption spectrum of enzyme bound FADH2, permitting the determination of dissociation constants and kinetics of binding.

Crystallization and preliminary x-ray investigation of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

P-Hydroxybenzoate hydroxylase (EC 1.14.13.2) can be crystallized from 0.1 M potassium phosphate buffer, pH 7.5, 39% saturated ammonium sulfate, and 1mM p-hydroxybenzoate. The space group is C2221 with 8 molecules/unit cell. When p-hydroxybenzoate is removed, the diffraction pattern and the cell dimensions change.

Intermediates in flavoprotein catalyzed hydroxylations

The reaction of molecular oxygen with the complex of reduced p-hydroxybenzoate hydroxylase and 2,4-dihydroxybenzoate has been followed by rapid reaction techniques. During the reaction, which produces stoichiometric amounts of oxidized enzyme and the hydroxylated product, 2,3,4-trihydroxybenzoate, three spectroscopically distinguishable intermediates have been detected and characterized.

P-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens: REACTIVITY WITH OXYGEN

Abstract p-Hydroxybenzoate hydroxylase, a flavoprotein isolated from Pseudomonas fluorescens, was anaerobically reduced and its reoxidation reactions with O2 studied with a stopped flow spectrophotometer. All effectors of this enzyme which stimulate its TPNH oxidase activity were found also to affect its reactivity with O2. Both the rates of reaction and the binding constant for O2 were enhanced by the effectors. During the reoxidation of the enzyme complexed with the substrate effector, p-hydroxybenzoate, a rapidly formed oxygenated-intermediate is observed. This intermediate is analogous to the oxygenated intermediate previously seen in the presence of 2,4-dihydroxybenzoate, another substrate effector (Spector, T., and Massey, V. (1972) J. Biol. Chem. 247, 5632–5636). It is believed that these intermediates are a hydroperoxide form of the flavin prosthetic group and are catalytic species that are produced prior to hydroxylation. When the reduced enzyme was reoxidized in its uncomplexed form, or complexed with a nonsubstrate effector (i.e. a compound which stimulates the TPNH oxidase activity but is not hydroxylated), no spectral intermediates were detected.

Kinetic studies on the enzyme-substrate complex formation of p-hydroxybenzoate hydroxylase by the stopped-flow method.

The spectrophotometric and spectrofluorometric investigations of the enzyme-substrate complex formation of p-hydroxybenzoate hydroxylase was made by the stopped-flow technique. The apparent velocity of the formation of the enzyme-substrate complex (the velocity of the absorbance change in visible and UV regions, and the velocity of the quenching of the fluorescence intensity in the FAD moiety of the holoenzyme by the substrate) was rapid enough to explain the maximal overall velocity (72 sec-1) or the activated anaerobic reduction rate (kred max= 200 sec-1). The results were consistent with a two-step mechanism involving a rapid bimolecular association of enzyme and substrate, and a slower follow-up unimolecular process.

P-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens: EVIDENCE FOR AN OXYGENATED FLAVIN INTERMEDIATE

Abstract The slow reaction of 2,4-dihydroxybenzoate (2,4-DOHB) with p-hydroxybenzoate hydroxylase in the presence of TPNH and oxygen, and the absence of auto-inhibition at high concentrations of 2,4-DOHB has facilitated a study of the hydroxylation reaction mechanism of this enzyme. The Vmax (45 min-1) of the over-all reaction and other kinetic constants were determined with steady state kinetics. Stopped flow spectrophotometric studies of the anaerobic reduction of the p-hydroxybenzoate hydroxylase-2,4-DOHB (E-S) complex by TPNH has demonstrated the formation of at least three intermediates during this process. All three species exhibit long wave length absorption bands but lack any detectable free radical EPR signals. Stopped flow studies on the reoxidation of the reduced E-S complex by oxygen have revealed the formation of a spectral intermediate that is believed to be an oxygenated form of the enzyme-bound flavin prosthetic group. A second intermediate tentatively identified as the oxidized enzyme-product complex is also detected during the reoxidation. This species decays at a rate similar to that of the Vmax; it is therefore likely that product dissociation is the over-all rate-limiting step.

Purification and Properties of p-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

The inducible p-hydroxybenzoate hydroxylase of Pseudomonas fluorescens which catalyzes the conversion of p-hydroxybenzoate to protocatechuate has been obtained in crystalline form as an electrophoretically homogenous protein which is very stable in the absence of added cofactors. The molecular weight is estimated to be 65,000. The enzyme contains 1 mole of flavin adenine dinucleotide per mole of protein. In the presence of p-hydroxybenzoate the enzyme-bound flavin is rapidly reduced by TPNH, but not by DPNH or reduced acetyl pyridine nucleotide. Under both anaerobic and aerobic conditions the enzyme can catalyze the rapid reduction of ferricyanide by TPNH. This rapid reaction requires the presence of the aromatic substrate. The absorption spectrum of the enzyme in the visible region is perturbed and the fluorescence of the enzymebound flavin is quenched in the presence of p-hydroxybenzoate. The binding of p-hydroxybenzoate to p-hydroxybenzoate hydroxylase follows 1:1 binding at concentrations of p-hydroxybenzoate less than 3 x 10-4 m . At higher concentrations, deviations from 1:1 binding are observed which correspond to the excess substrate inhibition observed in steady state kinetic analysis. A substrate analogue, 6-hydroxynicotinate, activates the enzyme with respect to reduction of the enzyme-bound flavin by TPNH, but is not hydroxylated during the subsequent reoxidation by molecular oxygen. During anaerobic reduction of the enzyme in the presence of p-hydroxybenzoate, two transient spectral intermediates are observable. These intermediates also occur during catalytic turnover. The intermediates are generated in sequential fashion. No electron paramagnetic resonance signals are associated with the transient spectral intermediates. The affinity of p-hydroxybenzoate hydroxylase for TPNH is enhanced at least 9-fold by p-hydroxybenzoate. The first order rate constant for the reduction of the enzyme-bound flavin by bound TPNH (i.e. kred) is increased 40,000-fold by p-hydroxybenzoate.

Studies on the Effector Specificity of p-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

Abstract 3,4-Dihydroxybenzoate (3,4-DOHB), 2,4-dihydroxybenzoate (2,4-DOHB), and benzoate increase the rate of TPNH oxidation by p-hydroxybenzoate hydroxylase and are therefore effectors of this enzyme. During the enzymatic reaction, in the presence of excess TPNH and O2, all of the 2,4-DOHB, l5% of the benzoate, and none of the 3,4-DOHB is hydroxylated. During the reactions with the non-hydroxylatable effectors, oxygen serves only to reoxidize the reduced flavin prosthetic group thereby producing hydrogen peroxide. No hydrogen peroxide is produced during the reactions with the hydroxylatable effectors (substrate effectors). All effectors cause significant perturbations in the absorbance, fluorescence, and circular dichroism spectra of the enzyme. Dissociation constants for the effectors from the enzyme complex were obtained from absorbance and fluorescence titrations. Studies on the nature of the intermediates produced during the static reduction of p-hydroxybenzoate hydroxylase in the presence and absence of the substrate effectors were performed with three different reductants. Small amounts of the neutral (blue) flavin semiquinone are produced during reduction in the absence of any effector and in the presence of 2,4-DOHB, but not in the presence of p-hydroxybenzoate (p-OHB). The free enzyme or the enzyme complexed with a substrate effector required 2.8 to 3.5 reducing eq per mole of flavin for complete reduction, but only donated 2.0 eq per mole of flavin during the reoxidation with potassium ferricyanide. The nature of the binding site at the active center was investigated with a number of p-OHB analogs. These compounds were found to be competitive inhibitors with respect to p-OHB. The inhibition and dissociation constants for some of the stronger inhibitors were determined.