Dihydrochelirubine 12monooxygenase

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Dihydrochelirubine 12-monooxygenase

Thalictrum bulgaricum enzyme, optimum pH 8.5, is a microsomal P450, specific for position 12. It requires oxygen and NAD(P)H [K776].

2.1.3 Hydroxylations of xenobiotics

Benzene hydroxylation

Rat liver mitochondrial enzyme, which requires NADPH, has a molecular weight of 52 000 [F686].

Methylococcus capsulatus methane monooxygenase (E.C. 1.14.13.25) hydroxylates benzene and halobenzenes by a NIH shift mechanism (products not stated) [F278].

Many other studies have also detected this reaction.

m-Cresol hydroxylase

Penicillium patulum enzyme acts on m-cresol to form methylquinol, as well as to form m -hydroxybenzyl alcohol. The enzyme, optimum pH 7.5, is a P450, which requires oxygen and NADPH, and is inhibited by carbon monoxide and cytochrome c. m-Cresol is a precursor of the antibiotic patulin, the formation of which requires both ring hydoxylation and side chain oxidation as well as ring fission and lactonisation [A1468].

Aniline hydroxylations

Aniline 0 o-aminophenol

This reaction has been detected in beef, mouse, pig, rat, sheep, rainbow trout, Boophilus and Nephila [A1795, A2788, A3460, D532, E60, J582].

Aniline 0 m-aminophenol

This reaction has been detected in Apocynum, Catharanthus and Conium [A2619].

Aniline 0 ^-aminophenol

Species in which activity has been found in liver microsomes include bandicoot, beef, bettong, kangaroo, man, monkey, mouse, pig, possum, quokka, rat, shrew, tree shrew, rainbow trout, Boophilus, Cunninghamella and Nephila [A1218, A1795, A1997, A2420, A3460, B145, D532, E60, J639]. In mammalia the reaction requires three factors for maximal activity: cytochrome P450 or P448, a reductase and a lipid component [A627, A735]. Cumene hydroperoxide can act as the oxidant [A335]. The activity of aniline-4-hydroxylase is not detectable in rat before birth. After birth, activity increases 30-fold to a maximum at weaning, and then declines until at six months it has decreased by 90 per cent [A71]. At birth, the activity is five times greater in female than in male rats, with higher activity at 10 than at five weeks age. The development pattern only follows the activity of P450 in broad outline [A736].

Besides P450, catalase, haemoglobin and myoglobin can catalyse this reaction [B940, D43, H614]. Human and sheep erythrocytes oxidize aniline to p-aminophenol, with NADPH as co-substrate; the enzyme appears to be oxyhaemo-globin [B64]. Human foetal haemoglobin is more active than adult haemoglobin [B145]. Other substrates hydroxylated are o - and m -toluidine and N-methylaniline, and associated reactions involve N- and O-demethylation [D135]. Ferrihaemoglobin a-chains are less active than b-chains [D283]. Methaemoglobin is also active [D43]. In haemoglobin and methaemoglobin hydroxylation of aniline, ascorbate and dihydrofumarate can replace NADH as electron donor [D557].

Chlorobenzene hydroxylation

Rat liver forms all three chlorophenols from chlorobenzene. Pre-treatment with 3-methylcholanthrene induces o -hydroxylation, whereas phenobarbital induces the formation of all three phenols [A2320].

Methylococcus capsulatus soluble methane monooxygenase (E.C. 1.14.13.25) hydroxylates chlorobenzene and a number of analogues by a NIH shift mechanism [F278].

Chlorophenol 4-monoxygenase

Burkholderia cepacia chlorophenol 4-monooxygenase not only catalyzes the formation of benzoates from benzaldehydes, but also the formation of o -hydroxybenzaldehydes, for instance with syringaldehyde as substrate. The reaction involves a NIH shift of the formyl group [K107].

2,4-Dichlorophenol hydroxylase (E.C. 1.14.13.20)

Acinetobacter enzyme is a homotetramer, molecular weight 240000 and optimum pH 7.6; it requires FAD and NADPH, but NADH is not so effective; FMN and riboflavin are inactive. It also acts on p -chlorophenol and 4-chloro-2-methylphenol, but some non-substrate chlorophenols induce the reduction of oxygen to peroxide [C232].

Proteobacteria enzyme, molecular weight 256 000, subunit molecular weight 65 000, pI 5.2 and optimum pH 8.0, requires NAD(P)H; the product is 3,5-dichlorocatechol. It has a broad specificity, but not for compounds in which both ortho positions are blocked [J466].

Pseudomonas cepacia enzyme is a homotetrameric flavoprotein, monomeric molecular weight 69 000 that requires FAD and NADPH; NADH is not so effective [G221].

4-Nitrophenol 2-monooxygenase (E.C. 1.14.13.29)

Nocardia enzyme, optimum pH 7.3, is soluble, requires oxygen, NAD(P)H and FAD. It is inducible, inactivated on dialysis and is somewhat unstable, even when frozen [D182].

5-Hydroxyisophthalate 4-hydroxylase

Bacterium enzyme, which is highly specific, is a flavoprotein containing FAD that cannot be replaced by FMN; it requires NAD(P)H [A2985].

Hydroxylation of phenoxyacetate

Aspergillus niger hydroxylates phenoxyacetate in o - and p -positions; oat and pea hydroxylate in the p -position. Arene oxides are postulated intermediates [A2849].

Biphenyl hydroxylations

Biphenyl 0 2-, 3- and 4-hydroxybiphenyl, and dihydroxybiphenyls

Hamster liver enzyme is a mixture of P450 and P448; 2-hydroxylation is induced preferentially by phenobarbital, and 3-methylcholanthrene induces 2- and 4- monohydroxylations [A323]. Carbon monoxide, SKF 525A and NADH inhibit 4-hydroxylation [A11].

Rat liver 2-hydroxylation (but not 3- and 4-hydroxylation) is induced by corticosteroids, especially by betamethasone. 4-Hydroxylation is, quantitatively, most important, and 3-hydroxylation the least [A3680]. All three monohydroxylations are induced by

3-methylcholanthrene [B195, A3680], and 3- and

4-hydroxylation by phenobarbital [A2371, A3680]. The reaction system involves P450, NADPH and NADPH-cytochrome c reductase; NADPH and reductase can be replaced by cumene hydroperoxide [A2371].

Rabbit intestinal and liver microsomal enzyme activity increases two- to four-fold from nine days after birth to the adult level [A1980].

Avocado mesocarp 2-hydroxylase is found in both microsomal and cytosolic fractions; microsomal activity is increased by pre-incubation with safrole or 3,4-benzpyrene. Activity in both fractions is only slightly inhibited by classical P450 inhibitors such as carbon monoxide or SKF 525A [A11].

Benzpyrene hydroxylases

The intense interest in this and related enzyme activities is consequential to the central role that 3,4-benzpyrene plays in the aetiology of human lung cancer.

Several publications have briefly reported on benzpyrene hydroxylase without specifying the site of hydroxylation. Many publications (see 3,4-Benzpyrene) report on 3-hydroxylation, but a significant number additionally report hydroxylation at positions 1 (rat, man and sole, e.g [A458, C473, J94]), 4 (rat [C204]), 7 (rat, man, mouse and scup, e.g. [A458, B122, D965, G445]), 8 (rainbow trout [A1424]) and 9 (rat, monkey, hamster, rabbit, mouse, man and Saccharomyces, e.g. [A458, A2327, A2379, A2814, A3326, A3412, A3681]). 3-Hydroxylation (E.C. 1.14.14.2) occurs in a large range of species, including man, rat [A13], monkey [A1869], tree shrew, pig [A1997], rabbit [A2729], camel [H103], quokka, kangaroo, bandicoot, [A2420], mouse [A3681], guinea pig [B82], pigeon, crow, kite, egret [C307], trout [A2145], goldfish, bullhead [D465], bluegill [E480], scup [D965], sole [G374], killifish [B259], mullet [C141], barnacle [B744], Saccharomyces [A3326] and Candida [B775].

Studies on monohydroxylation of benzpyrene and other polynuclear hydrocarbons is complicated by the ready dehydration of dihydrodiols under acid conditions to monophenols; the former are metabolic products formed by hydration of epoxides generated in metabolism of polynuclear hydrocarbons. In rat, it has been suggested that at least some 3-hydroxybenzpyrene is formed by a spontaneous rearrangement of benzpyrene-2,3-oxide [A3277].

In man, 3-hydroxylation has been described in bronchus [A13], placenta, [A114], lung [A1644], liver [A2379], blood [B122], hair follicle [B350], bladder [C138], hepatoma [E480], melanocyte [G360] and P450 isozymes [H10]. Placental enzyme is largely microsomal [A985].

In man and rat, liver enzyme is primarily microsomal, and is activated by low molecular weight cofactors. In man, the activity is twice as high in smokers compared with non-smokers. In rat, activity is three times higher in males than in females, but there are only small sex differences in man, rabbit and guinea pig [A313]. Rat liver microsomal enzyme activity is reduced by extraction with organic solvents [A331].

In rabbit, the liver enzyme activity remains low up to 16 days after birth, and then increases to or above the adult level at about 30 days [A1980].

2-Hydroxybiphenyl 3-monooxygenase

Pseudomonas azelaica enzyme, pI 6.3, is a homotetramer, molecular weight 256 000 and monomeric molecular weight 60 000, each monomer containing one mol of unbound FAD. It requires NADH and oxygen; the latter is incorporated into the substrate. Other 2-hydroxybiphenyls and substituted phenols are also substrates. Uncoupling of the reaction results in the formation of peroxide [E960, J392].

D-Amphetamine hydroxylation

Rat liver activity is mainly microsomal, but some is mitochondrial. Microsomal activity requires NADPH, with a fairly sharp optimum at pH 7.0 [A77].

Indole-3-butyrate 4-hydroxylation

Bupleurum and Phytolacca enzymes, molecular weights 10 000-12000 (both native and denatured) and optimum pH 5 /6, hydroxylate the indole nucleus [J838].

Carbostyril formation from quinolines

Rat and guinea pig liver aldehyde oxidase (E.C. 1.2.3.1) oxidize several N-alkylquinoliniums and analogues. N-Methyl-and N-phenylquinolinium both form the corresponding carbostyrils and 4-quinolones, and a similar reaction has been observed with N-methylphenanthridinium and N-methyl-5,6-benzoquinolinium [D147].

Comamonas testosteroni quinoline 2-oxidoreductase, molecular weight 360000, is composed of subunits, molecular weights 87 000, 32 000 and 22 000, and contains molybdenum, iron, acid labile sulphur, FAD and molybdopterin cytosine dinucleotide [K769].

Pseudomonas putida quinoline oxidoreductase, molecular weight 300 000 is composed of subunits, molecular weights 85 000, 30000 and 20 000. It contains eight Fe and two FAD/mol as well as molybdopterin cytosine dinucleotide. Other substrates are 5-, 6-, 7- and 8-hydroxyquinoline, and 8-chloroquinoline; the product from quinoline is carbostyril [F856, G151, K766].

Rhodococcus quinoline 2-oxidoreductase (E.C. 1.3.99.17), optimum pH 9.5, molecular weight 300 000, monomeric molecular weights 82000, 32000 and 18 000, contains molybdenum, iron, acid labile sulphur, FAD and molybdopterin cytosine dinucleotide. Further substrates include quinolines substituted with hydroxyl, methyl and chloro groups [K767].

This reaction has also been detected in Nocardia [E694] and Desulfobacterium [H917].

Quinaldine 4-oxidoreductase

Rat and guinea pig liver aldehyde oxidase forms 4-quinolones (see Carbostyril formation, above) [D147].

Arthrobacter enzyme, molecular weight 340 000 and monomeric molecular weights 82000, 35000 and 22000, contains molybdenum, iron and FAD. Molybdenum is present as molybdopterin cytosine dinucleotide. It forms 4(1H)-quinolones from several quinolines, and isoquinolines and analogues form the corresponding 1(2H)-oxo compounds. Aldehydes are also substrates [G803, J33].

Quinoline-4-carboxylate 2-oxidoreductase

An enzyme in Agrobacterium species, molecular weight 320 000, is composed of subunits, molecular weights 85 000, 35000 and 21 000. It contains eight mol of Fe, two Mo, and two FAD; molybdopterin cytosine dinucleotide is required for activity. Carbostyrils are formed from quinoline, 4-carboxyquinoline, 4-chloroquinoline and 4-methylquinoline [G778].

4-Hydroxyquinoline 3-monooxygenase

Pseudomonas putida enzyme is a trimer, molecular weight 126000. It requires oxygen and NADH, and it is specific for 1H-4-oxoquinoline [G812].

2-Hydroxyquinoline 8-monooxygenase

Pseudomonas putida enzyme is a highly specific two-component system. One is a yellow reduc-tase, molecular weight 38 000 which contains FAD and [2 Fe-2 S] units. It transfers electrons to an oxygenase, a homohexamer, monomeric molecular weight 55 000, and contains about six [2 Fe-2 S] units and additional Fe. The oxygenase requires the reductase, oxygen and NADH for activity, and activity is enhanced by polyethylene glycol and Fe2+ [K778].

Isoquinoline 1-oxidoreductase (E.C. 1.3.99.16)

Pseudomonas diminuta enzyme is dimeric, monomeric molecular weight 16 000 and 80 000, and pI in the range 6.2 /6.8. It contains molybdenum, iron, acid labile sulphur, phosphate and cytosine monophosphate (probably as molybdopterin cytosine dinucleotide), but no FAD. It requires an electron acceptor, but not oxygen or NAD, and acts on isoquinoline and quinazoline (to form 1- and 4-oxo compounds, respectively) as well as analogues; quinolines are not substrates [K765]. DNA sequencing indicates monomeric molecular weights of 16 399 and 84249 for the monomers [K764].

Debrisoquine 4-hydroxylase

Human enzyme is microsomal, requiring NADPH. It is not found in all human subjects [C324].

Rat liver microsomal enzyme has been purified as a specific P450. It also N- and O-dealkylates other compounds [D98].

Hydroxylation with internal hydroxyl transfer

Horse liver alcohol dehydrogenase (E.C. 1.1.1.1) acts on 2-hydroxylaminofluorene to form 2-amino-1- and 3-hydroxyfluorene [H291]. Comamonas enzyme reduces 1-chloro-

4-nitrobenzene anaerobically to 2-amino-

5-chlorophenol, suggesting that there is an initial reduction to the hydroxylamine, followed by a Bamberger rearrangement, with hydroxyl migration to form the final product [K80].

2.1.4 Hydroxylation with elimination of substituent

4-Hydroxybenzoate 1-hydroxylase (decarboxylating) (E.C. 1.14.13.64)

Candida parapsilosis enzyme is a monomer, molecular weight 50 000 and optimum pH 8 containing FAD, which requires oxygen and NAD(P)H. Quinol is formed, with molecular oxygen incorporated into the new hydroxyl group. It also acts on a range of ring-substituted 4-hydroxybenzoates [J461].

This reaction has also been found in Pycnoporus cinnabarinus [D569].

Gentisate 1-hydroxylase (decarboxylating)

hydroxyquinol with the release of carbon dioxide [E288]

Salicylate 1-monooxygenase (E.C. 1.14.13.1)

e.g. Salicylate 0 catechol + CO2

Pseudomonas putida enzyme is a monomer, molecular weight 54 000. The amino acid composition and the terminal amino acids have been determined [A1216]. Another study using a different strain found a molecular weight of 45 000, with a different amino acid composition [F658]. The reaction requires oxygen and NADH, with carbon dioxide as the second product. Salicylaldehyde is also a substrate, and this releases formate [B751]. By using specifically ring-labelled salicylate it has been found that decarboxylation and hydroxylation with P. cepacia enzyme occur at the same carbon atom [B883]. It is an inducible enzyme [E287]. A Pseudomonas enzyme also acts on 3- and 5-chlorosalicylate and 3,5-dichlorosalicylate [H506].

Trichosporon cutaneum enzyme, molecular weight 45 300 and optimum pH 7.5, contains FAD. It also acts on salicylates substituted with a hydroxyl group at positions 3, 4, 5 and 6, an amino or chloro group at 4 or 5, a methyl at 4, or a methoxy or fluoro at 5 [D232].

This reaction has been found to occur non-enzymatically in rat, catalysed by free radicals formed by the parkinsonism-inducing ion MPP + [J681]. In rat liver the formation of catechol and other products from salicylate is catalysed by the action of hydroxyl free radicals rather than by the direct action of a decarboxylating hydroxylase [J754].

Trichosporon cutaneum acts on gentisate to form hydroxyquinol [B368].

3,4-Dihydroxybenzoate 1-hydroxylase (decarboxylating)

Trichosporon cutaneum enzyme is highly specific. It requires oxygen and NADH, and forms

Vanillate 1-hydroxylase (decarboxylating)

Sporotrichum pulverulentum enzyme acts on vanillate, protocatechuate, gallate, 2,4-dihydroxybenzoate and p -hydroxybenzoate. Homovanillate and 2,3,4-trihydroxybenzoate are less effective as substrates, whereas gentisate, syringate, ferulate, veratrate and p -methoxy-

4-Hydroxyisophthalate hydroxylase benzoate are poor substrates, and benzoate and m -methoxybenzoate are not substrates. The product from vanillate is methoxyquinol [B73]. The enzyme has a molecular weight of 65 000 and requires NADPH and FAD for maximal activity. Tiron, Cu2 +, Ag +, Hg2 + and p -chloromercuri-benzoate are inhibitory, whereas EDTA, diethyldithiocarbamate and Fe3+ are not. Further substrates are 3,4-dihydroxy-5-methoxybenzoate and 2,4,6-trihydroxybenzoate [C57].

Phanerochaete chrysosporium enzyme, optimum pH 7.5-8.5, requires NAD(P)H and oxygen, and is cytosolic [B312].

4-Hydroxyisophthalate hydroxylase

Pseudomonas enzyme, which is different from p -hydroxybenzoate hydroxylase, is a homodimer, molecular weight 103 000, containing one mol of FAD, with protocatechuate as the reaction product. It also acts on 5-sulphosalicylate at a much lower rate; other compounds are not substrates. It is inhibited by substrate analogues and thiol-binding compounds, and is stabilized by thiols [A3066].

Aniline oxidation to catechol some conditions 3-hydroxyanthranilate is also formed; this requires a reducing agent that acts on imines [C393, E270].

Aspergillus niger, molecular weight 44 000, pI 5.36 and optimum pH 8.2, requires FAD and Fe2+. It is inhibited by Cu , o-phenanthroline and p -chloromercuribenzoate. Another activity associated with this enzyme is anthranilate 3-hydroxylation [A778, B327, D231].

This reaction has also been observed in Aspergillus soyae [A1299].

Anthranilate hydroxylase (with deamination and decarboxylation; E.C. 1.14.12.1)

Pseudomonas cepacia 2-halobenzoate 1,2-dioxygenase acts on anthranilate and other benzoates with a considerable range of substituents [G434].

Trichosporon cutaneum enzyme, molecular weight 94000 and optimum pH 7.7, appears to be a dimer. Substrates are anthranilate and N-methylanthranilate; some benzoates are non-substrate effectors, with the formation of peroxide from oxygen [C393].

This reaction has been observed in Micrococcus, Aspergillus soyae and Pseudomonas pyrrocinia [A1299, C120, D56].

Nocardia carries out this reaction, with the incorporation of molecular oxygen; a cyclic peroxide intermediate has been suggested [A1520].

Anthranilate 3-monooxygenase (deaminating)

Anthranilate 0 pyrocatechuate

Trichosporon cutaneum enzyme, molecular weight 95 000 and monomeric molecular weight 50 000, contains two mol of FAD and exhibits a sharp optimum pH at 7.7. The oxygen in position 2 comes from water, and in position 3 from molecular oxygen. Other substrates are N-methyl- and N,N-dimethylanthranilate. Under

4-Aminobenzoate hydroxylase (E.C. 1.14.13.27)

4-Aminobenzoate 0 ^-aminophenol + CO2

Agaricus bisporus enzyme, molecular weight 49000, pI 6.0 and optimum pH 6-8 (partly dependent on cofactor), contains about one mol of FAD. It requires oxygen and NAD(P)H. Other substrates include aminobenzoates and p -hydroxybenzoate. Unlike 4-aminobenzoate, these substrates also form peroxide non-stoichiometrically, whereas peroxide is formed stoichiometrically relative to cofactor oxidation in the presence of other benzoates that are not substrates. Neither FMN nor riboflavin can replace FAD [D770, E162].

2-Aminobenzenesulphonate 2,3-dioxygenase

2-Aminoben/enesulphonate 2,3-dioxygenase

Alcaligenes enzyme is monomeric, molecular weight 42 000, and requires two oxygen molecules to form 2,3-dihydroxybenzenesulphonate. Maximal activity is found near the end of the exponential growth phase [H239, K775].

Alcaligenes enzyme, molecular weight 134000 according to another study, is composed of two pairs of monomers, monomeric molecular weights 45 000 and 16000, with one [2Fe-2S] centre associated with each of the larger chains. Inhibition by o -phenanthroline indicates the presence of another Fe-binding site. The N-terminal sequences have been determined. The product formed from 2-aminobenzenesulphonate is 2,3-dihydroxybenzenesulphonamide. Other substrates are benzenesulphonate, and benzene-sulphonates substituted with nitro, amino, chloro and hydroxyl groups; the reaction products from these compounds were not identified [K293].

4-Sulphobenzoate 3,4-dioxygenase

Comamonas testosteroni contains two isozymes. One is a red dimer, monomeric molecular weight 50 000, and the other a yellow monomer, molecular weight 36000, with NADH and Fe2+ as cofactors. The enzyme is highly specific, forming sulphite and protocatechuate. The reaction does not appear to involve two steps or to involve an intermediate dihydrodiol [G244].

Pseudomonas putida oxidizes benzenesulpho-nate to catechol and p-toluenesulphonate to 4-methylcatechol [J657]. The same reaction is observed in Alcaligenes [H297].

Salicylaldehyde hydroxylation

Pig liver microsomal flavin-containing monooxygenase-1 forms catechol and formate in equimolecular amounts from salicylaldehyde [H482].

Dechlorination with concomitant hydroxylation by microorganisms a. 4-Chlorobenzoate dehalogenase (E.C. 3.8.1.6)

^-Halobenzoate 0 ^-hydroxybenzoate

Studies have found that this reaction occurs in three stages: conjugation with CoA (E.C. 6.2.1.33) followed by dehalogenation (E.C. 3.8.1.7) and hydrolysis (E.C. 6.2.1.33) [K191]; detailed information is found under these headings.

An Arthrobacter enzyme, optimum pH 6.8, is activated by Mn2+ and inhibited by peroxide. Other substrates are p-fluoro- and p -bromobenzoates [D544]. Another study claims that although p -iodobenzoate is a substrate p -fluorobenzoate is not, nor are p -chlorophenylacetate or p -chlorocinnamate. The molecular weight is about 45 000 and the optimum pH 7-7.5. Unlike all other similar dehydrogenases reported at the time it is not inhibited by EDTA or activated by Mn2+ [E752].

A Pseudomonas dehalogenase requires ATP, CoA and Mg2+ [G205]. The incorporated oxygen comes entirely from water, indicating that molecular oxygen is not involved [D473] (but see b. below). This enzyme, optimum pH 7-7.5, is activated by Mn2+ or Co2 + , and is inhibited by EDTA. It acts on chloro-, bromo- and iodobenzoates, but not on p -fluorobenzoate [E359].

b. p-Chlorobenzoyl CoA dehalogenase (E.C. 3.8.1.7)

^-Chlorobenzoyl CoA 0 p-hydroxybenzoyl CoA

This is the second step in the reaction sequence leading to the oxidative dehalogenation of p -chlorobenzoate.

Pseudomonas 4-chlorobenzoyl CoA dehalogenase also acts on the bromo- and iodo- analogues, but not on the fluoro- analogue [G920, H645]. About 75 per cent of the incorporated oxygen comes from molecular oxygen, and the remainder from water [H216]. It is a homotetramer with high temperature

Dechlorination with catechol formation stability, molecular weight 120000, pI 6.7 and optimum pH 10 [H645, K191].

Acinetobacter enzyme also acts on bromo-analogues [G896].

Arthrobacter enzyme is a tetramer, monomeric molecular weight 33 000, pI 6.1 and optimum pH 8, with a stability range pH 6.5-10. It also acts on the fluoro-, bromo- and iodo- analogues, but not on o - and m - chloro analogues [H279].

c. Chlorophenol 4-monooxygenase

Burkholderia enzyme is a two-component system. One is a reductase, molecular weight 22 000 that contains FAD, and the other has a molecular weight of 58 000. The reaction requires oxygen and NADH. 2,4,5-Trichlorophenol is oxidized to 2,5-dichloroquinol and then to 5-chloro-2-hydroxyquinol. Other substrates are 2,3,5,6-tetrachlorophenol, 2,4,6-trichlorophenol and 2,5-dichlorophenol [J177].

Dechlorination with catechol formation a. 2-Chlorobenzoate 1,2-dioxygenase (E.C. 1.14.12.13)

Pseudomonas cepacia forms pyrocatechuate from 2-chlorobenzoate [F401].

b. 2-Halobenzoate 1,2-dioxygenase o-Halobenzoate 0 catechol

Pseudomonas cepacia enzyme is composed of two protein fractions, and acts on a range of o -halobenzoates as well as other o -substituted benzoates. In many instances the products have not been identified, but benzoate, o -toluate, m -hydroxybenzoate and p -hydroxybenzoate form benzoate-1,2-dihydrodiol, o -cresol, gentisate and quinol respectively; in none of these compounds is there a readily displaceable ortho substituent, which drives the reaction in a different direction [G434].

c. 4-Chlorophenylacetate 3,4-dioxygenase (E.C. 1.14.12.9)

^-Chlorophenylacetate 0 3,4-dihydroxyphenylacetate

This Pseudomonas activity involves a two-component, intensely red-brown enzyme system. One of the enzymes is a homotrimer, molecular weight 140000 and pI 5.0. In the reaction two oxygen atoms are incorporated from molecular oxygen [E163].

d. 2,4,6-Trichlorophenol 4-monooxygenase

Azotobacter enzyme is a homotetramer, molecular weight 240000, which forms 2,6-dichloroquinol from 2,4,6-trichlorophenol. It requires FAD, and utilizes one mol of oxygen and two mol of NADH. Other substrates include a range of para -chlorinated or brominated phenols; o -chlorophenol is a poor substrate [H884].

Pentachlorophenol monooxygenase

Arthrobacter enzyme, optimum pH 7.5, is stimulated by EDTA and requires NADPH and oxygen, with formation of tetrachloroquinol. Other substrates include 2,3,4- 2,4,5-, and 2,4,6-trichlorophenol, and 2,3,4,5-tetrachloro-phenol, but other analogues are not substrates [K750].

A Flavobacterium enzyme, molecular weight 63 000, pI 4.3 and optimum pH 7.5-8.5, can exist as polymeric forms. The monomer is a flavoprotein containing one mol of FAD, and the reaction requires two mol of NADPH. It hydroxylates pentachlorophenol to tetrachloroquinol [G272].

Sphingomonas pentachlorophenol 4-monooxygenase, which requires NADPH, is a flavoprotein, molecular weight predicted to be 59993 with 538 amino acid residues by DNA studies. Other substrates include 4-nitrocatechol and p -nitrophenol [K73].

Quinol formation from para substituted phenols

Rat liver microsomes form quinol from phenols substituted in the para position with nitro, cyano, hydroxymethyl, acetyl, benzoyl or halide groups. Results from experiments using 18O2 suggest that

Defluorination with catechol formation the initial reaction is the formation of a 4-hydroxy-1-oxo-2,5-diene, involving oxygen transport by the Fe in the enzyme. This intermediate may then either form quinol directly, by elimination of the substituent (as a positively charged ion) from the 4-position, or the formation of p -quinone, with the substituent forming a negatively charged ion. Trapping experiments suggest that both mechanisms may occur, depending on the identity of the substituent. In the case of p -cresol the side chain is not eliminated; instead there is a NIH shift to form toluquinol [J81].

Defluorination with catechol formation

Pseudomonas converts 3-fluorinated compounds into the corresponding 2,3-dihydroxy analogues. Although fluorobenzene is not a substrate, toluene, anisole, fluorobenzene, chlorobenzene, bromobenzene, iodobenzene, benzonitrile and benzyl alcohol fluorinated at position 3 are substrates [F76].

2-Nitrotoluene 2,3-dioxygenase

2-Nitrotoluene + O20 3-methylcatechol + nitrite

Pseudomonas enzyme is a three-component system which forms 3-methylcatechol as well as o -nitrobenzyl alcohol from 2-nitrotoluene (the best substrate). A series of other nitrotoluenes and nitrobenzene are also catechol-forming substrates. Both oxygen atoms are incorporated from molecular oxygen [H344, J524].

2-Nitrophenol 2-monooxygenase (E.C. 1.14.13.31)

o-Nitrophenol + O2 0 catechol + nitrite

Pseudomonas forms catechol and nitrite from o -nitrophenol [E726]. P. putida enzyme, molecular weight 58 000-65000, optimal activity and optimum stability at pH 7.5-8.0, requires NADPH and oxygen. It is activated by Mg2+ or Mn2 +, but not by flavins. It is very sensitive to heat inactivation, but is stabilized by substrate. It also acts on a range of other o -nitrophenols, but not all o -nitrophenols are substrates [E607, H65, K898].

2,4-Dinitrotoluene 3,4-dioxygenase

2,4-Dinitrotoluene + O2 0

4-methyl-5-nitrocatechol + nitrite

A Pseudomonas enzyme catalyzes this reaction [G407, J524].

p -Nitrophenol hydroxylation

^-Nitrophenol + O2 0 quinol + nitrite

Penicillium chrysogenum enzyme is found in a membrane fraction. It requires oxygen and NAD(P)H, and is stimulated by FAD; the oxygen atom is incorporated from molecular oxygen [E94].

Moraxella forms quinol and nitrite from p -nitrophenol [G153].

4-Nitrocatechol hydroxylation

Sphingomonas pentachlorophenol 4-monooxygenase (see above) oxidizes 4-nitrocatechol to hydroxyquinol, and p -nitrophenol (poorly) to quinol, with the release of nitrite [K73].

Bacillus sphaericus and Arthrobacter enzymes form hydroxyquinol with the release of nitrite [H270, J679].

6-Nitrobenzo[« ]pyrene hydroxylation

Rat lung and liver catalyse the conversion of 6-nitrobenzo[« ]pyrene into 6-hydroxybenzo[« ]pyrene, also with the formation of a trace of benzpyrene [E467]. The same reaction has been observed in mouse [E971].

2.2 Formation of quinones and analogues from catechols, quinols and other precursors

Monophenol monooxygenase (E.C. 1.14.18.1)

Tyrosinase (classically from mushroom or skin melanocytes), polyphenol oxidase (from potato) and laccase (E.C. 1.10.3.2; originally from the lacquer tree) are old classifications for enzymes that are now grouped together under the heading of Monophenol Monooxygenase, although these classifications are still commonly found in the literature. In the current section, this classification (except polyphenol oxidase) has been used because it is still in such common use. They are all copper-containing enzymes, but they differ in specificity; laccases preferentially oxidize p -phenylenediamine relative to oxidation of catechols to quinones, but they differ from tyrosinase in that they do not oxidize tyrosine to dopa. However, the distinctions between these classes of enzyme are not clear-cut.

One study with o - and p -diphenol oxidases (considered to be catecholases and laccases respectively) from apple, banana, Agaricus, Glomerella, Sclerotina (o -oxidases), peach, spruce, Botrytis, Coriolus, Trametes, Glomerella and Rhus (p -oxidases) distinguishes between the activites on the basis of the following parameters:

Some distinguishing features between catechol oxidase and laccase

Test

Catechol oxidase

Laccase

Catechols

Oxidized

Oxidized

Quinols

Zero or poor

Oxidized

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