Flavoprotein Hydroxylases Research Articles

Purification and Characterization of 2-Hydroxybiphenyl 3-Monooxygenase, a Novel NADH-dependent, FAD-containing Aromatic Hydroxylase from Pseudomonas azelaica HBP1

2-Hydroxybiphenyl 3-monooxygenase (HbpA), the first enzyme of 2-hydroxybiphenyl degradation in Pseudomonas azelaica HBP1, was purified 26-fold with a yield of 8% from strain HBP1 grown on 2-hydroxybiphenyl. The enzyme was also purified from a recombinant of Escherichia coli JM109, which efficiently expressed the hbpA gene. Computer densitometry of scanned slab gels revealed a purity of over 99% for both enzyme preparations. Gel filtration, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the enzyme was a homotetramer with a molecular mass of 256 kDa. Each subunit had a molecular mass of 60 kDa containing one molecule of noncovalently bound FAD. The monooxygenase had a pI of 6.3. It catalyzed the NADH-dependent ortho-hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl. Molecular oxygen was the source of the additional oxygen of the product. The enzyme hydroxylated various phenols with a hydrophobic side chain adjacent to the hydroxy group. All substrates effected partial uncoupling of NADH oxidation from hydroxylation with the concomitant formation of hydrogen peroxide. 2,3-Dihydroxybiphenyl, the product of the reaction with 2-hydroxybiphenyl, was a non-substrate effector that strongly facilitated NADH oxidation and hydrogen peroxide formation without being hydroxylated and also was an inhibitor. The apparent Km values (30 degrees C, pH 7.5) were 2.8 microM for 2-hydroxybiphenyl, 26.8 microM for NADH, and 29.2 microM for oxygen. The enzyme was inactivated by p-hydroxymercuribenzoate, a cysteine-blocking reagent. In the presence of 2-hydroxybiphenyl, the enzyme was partly protected against the inactivation, which was reversed by the addition of an excess of dithiothreitol. The NH2-terminal amino acid sequence of the enzyme contained the consensus sequence GXGXXG, indicative of the betaalphabeta-fold of the flavin binding site and shared homologies with that of phenol 2-hydroxylase from Pseudomonas strain EST1001 as well as with that of 2,4-dichlorophenol 6-hydroxylase from Ralstonia eutropha.

Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

The gene encoding 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO; EC 1.14.12.4) was cloned by using an oligonucleotide probe corresponding to the N terminus of the enzyme to screen a DNA library of Pseudomonas sp. MA-1. The gene encodes for a protein of 379 amino acid residues corresponding to a molecular mass of 41.7 kDa, the same as that previously estimated for MHPCO. MHPCO was expressed in Escherichia coli and found to have the same properties as the native enzyme from Pseudomonas sp. MA-1. This study shows that MHPCO is a homotetrameric protein with one flavin adenine dinucleotide bound per subunit. Sequence comparison of the enzyme with other hydroxylases reveals regions that are conserved among aromatic flavoprotein hydroxylases.

Unusual mechanism of oxygen atom transfer and product rearrangement in the catalytic reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

The oxygenation reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with the substrate, MHPC, was investigated. Two oxygenated flavin intermediates C(4a)-hydroperoxy flavin and C(4a)-hydroxy flavin were found, implying that the enzyme functions similarly to flavoprotein hydroxylases. This finding is supported by the results of independent oxygen-18 tracer experiments, which showed that one atom of oxygen from 18O2 and one atom of oxygen from H218O are incorporated in the product. MHPC oxygenase normally catalyzes both the oxygenation and the hydrolytic ring opening of the pyridine ring of MHPC to yield the acyclic compound, alpha-(N-acetylaminomethylene)succinic acid. Using 5-hydroxynicotinic acid (5HN), which has no 2-methyl group, we tested whether the hydrolytic reaction was due to the presence of the 2-methyl group on MHPC (that prevented rearomatization of the initial product) or to the specific properties of MHPC oxygenase. Product analysis of the enzymatic reaction of 5HN and MHPC oxygenase shows that the enzyme catalyzes the hydroxylation and subsequent hydrolysis of the hydroxylated substrate to yield an acyclic product. The investigation of the oxygenation reaction demonstrates that the enzyme uses the same mechanism to catalyze the 5HN reaction as it does in the MHPC reaction.

Complete nucleotide sequence of tbuD, the gene encoding phenol/cresol hydroxylase from Pseudomonas pickettii PKO1, and functional analysis of the encoded enzyme.

The gene (tbuD) encoding phenol hydroxylase, the enzyme that converts cresols or phenol to the corresponding catechols, has been cloned from Pseudomonas pickettii PKO1 as a 26.5-kbp BamHI-cleaved DNA fragment, designated pRO1957, which allowed the heterogenetic recipient Pseudomonas aeruginosa PAO1c to grow on phenol as the sole source of carbon. Two subclones of pRO1957 carried in trans have shown phenol hydroxylase activity in cell extracts of P. aeruginosa. The nucleotide sequence was determined for one of these subclones, a 3.1-kbp HindIII fragment, and an open reading frame that would encode a peptide of 73 kDa was found. The size of this deduced peptide is consistent with the size of a novel peptide that had been detected in extracts of phenol-induced cells of P. aeruginosa carrying pRO1959, a partial HindIII deletion subclone of pRO1957. Phenol hydroxylase purified from phenol-plus-Casamino Acid-grown cells of P. aeruginosa carrying pRO1959 has an absorbance spectrum characteristic of a simple flavoprotein; moreover, the enzyme exhibits a broad substrate range, accommodating phenol and the three isomers of cresol equally well. Sequence comparisons revealed little overall homology with other flavoprotein hydroxylases, supporting the novelty of this enzyme, although three conserved domains were apparent.

Studies of the oxidative half-reaction of anthranilate hydroxylase (deaminating) with native and modified substrates.

The oxidative half-reactions of anthranilate hydroxylase (EC 1.14.12.2) were examined in the presence of anthranilate and modified substrates. C(4a)-Hydroperoxyflavin (C(4a)-FlOOH) and C(4a)-hydroxyflavin (C(4a)-FlOH) intermediates were detected in oxidative reactions with all substrates. Thus, the oxygenation reactions of the enzyme are similar to those of flavoprotein hydroxylases that convert phenolic compounds to catechols. These observations support a mechanism proposed for this enzyme (Powlowski, J. B., Dagley, S., Massey, V., and Ballou, D. P. (1987) J. Biol. Chem. 262, 69-74) involving nucleophilic attack of the substrate on C(4a)-FlOOH, and formation of an imine intermediate that is subsequently hydrolyzed. Anthranilate hydroxylase is therefore a typical flavoprotein hydroxylase with the added capacity of hydrolyzing imine intermediates. Fluorine substituents on the aromatic ring decreased the rate of conversion of C(4a)-FlOOH to C(4a)-FlOH, as predicted by this mechanism. Hydroxylation of 3-fluoro- and 3-methylanthranilates resulted in the formation of nonaromatic products that appeared to stabilize the C(4a)-FlOH. No evidence was found for a high extinction intermediate (intermediate II) (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563) under conditions where it was readily detected with other flavoprotein hydroxylases. It was shown that the spectra of the nonaromatic products (which are quinonoid forms) could not be summed with the spectra of C(4a)-hydroxyflavin to obtain that of a putative intermediate II, thus ruling out that explanation for previous observations of II.

Absorption spectra of the hydroxycyclohexadienyl radicals of substrates for phenol hydroxylase.

The absorption spectra of the hydroxycyclohexadienyl radicals formed upon the addition of OH radicals to six substrates for phenol hydroxylase have been determined using pulse radiolysis. Combining the radical spectra of thiophenol (lambda max, 390 nm; epsilon, 10,500 M-1 cm-1) and resorcinol (lambda max, 340 nm; epsilon, 4,100 M-1 cm-1) with their respective published spectra of enzyme-bound reduced flavin that is substituted in the C(4a) position of the dihydroflavin ring gave composite spectra that closely match the spectra formed concomitantly with the introduction of an oxygen atom into the substrates, the so-called Intermediate II species. A similar procedure for the substrates hydroquinone, 3-aminophenol, 3-chlorophenol, and 3-methylphenol yielded spectra that are also consistent with the known characteristics of their Intermediate II species. These spectral results give further support to the proposed biradical mechanism (Anderson, R.F., Patel, K. B., and Stratford, M. R. L. (1987) J. Biol. Chem. 262, 17475-17479) for the functioning of this class of flavoprotein hydroxylases.

Regulation of oxidation-reduction potentials of anthranilate hydroxylase from Trichosporon cutaneum by substrate and effector binding.

The pH dependence of the redox behavior of anthranilate hydroxylase from Trichosporon cutaneum in its uncomplexed and anthranilate-complexed forms, as well as the effects on the reduction potential, at pH 7.4, of enzyme in complex with 3-methylanthranilate, salicylate, 3-acetylpyridine adenine dinucleotide phosphates, and azide plus anthranilate, is described. At pH 7.4 the midpoint potential of uncomplexed enzyme (EFlox/EFlredH-) is -0.229 V vs SHE, close to that of free flavin. The aromatic substrates and effector all shift the midpoint potential value in a positive direction by 0.068-0.100 V. This shift results in thermodynamically more favorable reduction of the substrate/effector-complexed enzyme by NADPH. Consistent with thermodynamic considerations, the aromatic substrates (or effector) are bound to the reduced enzyme 2-4 orders of magnitude more tightly than to the oxidized enzyme. The tighter binding of the substrate to the two-electron-reduced enzyme may be related to the double hydroxylation reaction performed by this enzyme, which is a more complex reaction than is carried out by typical flavoprotein hydroxylases. The acetylpyridine nucleotides appear to have no significant regulatory role.

Absorption spectra of radicals of substrates for p-hydroxybenzoate hydroxylase following electrophilic attack of the .OH radical in the 3 position.

The spectra of radicals formed upon the addition of .OH radicals to the 3 position of substrates for p-hydroxybenzoate hydroxylase (4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and 4-aminobenzoic acid) have been determined using pulse radiolysis combined with the results from high performance liquid chromatography measurements. The 3-hydroxy radical forms of the substrates absorb maximally in the 365-410 nm region with extinction coefficients in the range 3700-5250 M-1 cm-1. Upon combining these radical spectra with the known spectrum of enzyme-bound reduced flavin that is substituted in the C(4a) position of the isoalloxazine ring, spectra are found which closely resemble the species long thought to be formed concomitantly with the introduction of an oxygen atom into substrates. On the basis of these spectral results a new radical mechanism is proposed for the functioning of this class of flavoprotein hydroxylases.

8-Mercaptoflavins as active site probes of flavoenzymes.

Representative examples of the various classes of flavoproteins have been converted to their apoprotein forms and the native flavin replaced by 8-mercapto-FMN or 8-mercapto-FAD. The spectral and catalytic properties of the modified enzymes are characteristically different from one group to another; the results suggest that flavin interactions at positions N(1) or N(5) of the flavin chromophore have profound influences on the properties of the flavoprotein. 1. The 8-thiolate anion form of 8-mercaptoflavin has an absorption maximum in the region 520 to 550 nm epsilon approximately 30 mM-1 cm-1). This form is retained on binding to flavoproteins whose physiological reactions involve obligatory one-electron transfers (e.g. flavodoxin, NADPH-cytochrome P-450 reductase). In the native form these enzymes stabilize the blue neutral radical of the flavin. A radical form of 8-mercaptoflavin is also stabilized by these proteins. 2. The p-quinoid form of 8-mercaptoflavin has an absorption maximum in the range 560 to 600 nm (epsilon approximately 30 mM-1 cm-1). This form is stabilized on binding to flavoproteins of the dehydrogenase-oxidase class (e.g. glucose oxidase, D-amino acid oxidase, lactate oxidase, Old Yellow Enzyme). These same enzymes in their native flavin form stabilize the red semiquinone, and have a pronounced reactivity with sulfite to form flavin N(5)-sulfite adducts. These properties of the native enzyme, including the ability to react with nitroalkane carbanions, are not exhibited by the 8-mercaptoflavoproteins. 3. A group of flavoenzymes fails to conform strictly to the above classification, exhibiting some properties of both classes. These include the examples of flavoprotein hydroxylases and transhydrogenases studied. 4. The riboflavin-binding protein of hen egg whites binds 8-mercaptoriboflavin preferentially in the unionized state, resulting in a shift in pK from 3.8 with free 8-mercaptoriboflavin to greater than or equal to 9.0 with the protein-bound form.

52] Salicylate hydroxylase

Publisher Summary This chapter provides information on enzyme salicylate hydroxylase. Salicylate hydroxylase is a flavoprotein hydroxylase, a member of a class of enzymes elaborated by some soil bacteria, typically pseudomonads. These enzymes are induced by the presence of phenolic compounds. The activity of the enzyme was determined spectrophotometrically by following the rate of substrate-dependent oxidation of NADH in a Cary 14 or Gilford 2400 spectrophotometer at 340 nm. Salicylate hydroxylate is established as a flavoprotein containing flavin adenine dinucleotide (FAD) as the sole prosthetic group. The purified hydroxylase migrates as one homogeneous and symmetrical peak in the Model E analytical untracentrifuge with 0.1 M KCl as solvent. Enzyme activity can also be assayed by measuring the rate of oxygen consumption using an oxygen electrode. This enzyme, in addition to its FAD prosthetic group, has three substrates: salicylate or other aromatic compound, pyridine nucleotide, and oxygen. Its kinetics, therefore, can be expected to be complex.

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.

Mechanism of aromatic hydroxylation: Properties of a model for pyridine nucleotide-dependent flavoprotein hydroxylases

A model (NADH-phenazine methosulfate-O 2) formally similar to pyridine nucleotide-dependent flavoprotein hydroxylases catalyzed the hydroxylation of several aromatic compounds. The hydroxylation was maximal at acid pH and was inhibited by ovine Superoxide dismutase, suggesting that perhydroxyl radicals might be intermediates in this process. The stoichiometry of the reaction indicated that a univalent reduction of oxygen was occurring. The correlation between the concentration of semiquinone and hydroxylation, and the inhibition of hydroxylation by ethanol which inhibited semiquinone oxidation, suggested the involvement of phenazine methosulfate-semiquinone. Activation of hydroxylation by Fe 3+ and Cu 2+ supported the contention that univalently reduced species of oxygen was involved in hydroxylation. Catalase was without effect on the hydroxylation by the model, ruling out H 2O 2 as an intermediate. A reaction sequence, involving a two-electron reduction of phenazine methosulfate to reduced phenazine methosulfate followed by disproportionation with phenazine methosulfate to generate the semiquinone, was proposed. The semiquinone could donate an electron to O 2 to generate O 2 which could be subsequently protonated to form the perhydroxyl radical.

3-Hydroxybenzoate 6-hydroxylase from [formula omitted

An inducible 3-hydroxybenzoate 6-hydroxylase has been purified to homogeneity from Pseudomonas aeruginosa . It contains FAD as a prosthetic group. 3-Hydroxybenzoate is quantitatively hydroxylated to give gentisate with equimolar consumptions of NADH and O 2. NADPH will substitute as an electron donor, and several aromatic analogues of 3-hydroxybenzoate stimulate reduced nucleotide oxidation by the enzyme with formation of both hydrogen peroxide and hydroxylated products. Of various analogues of 3-hydroxybenzoate, those substituted in 2,4,5 and 6-positions are competent substrates; partial uncoupling of electron flow from hydroxylation with concomitant formation of hydrogen peroxide and “gentisates” occurs. The “natural” product of the reaction, gentisate, is an effector in that it stimulates NADH oxidation with the formation of hydrogen peroxide. 3-hydroxybenzoate 6-hydroxylase thus resembles other flavoprotein hydroxylases in the general regulatory properties dictated by their aromatic substrates, pseudosubstrates or effectors.