Formation and degradation of sidechains

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4.1 Side-chain formation

Nuclear C-methyl incorporation

1. Benzene and toluene

Human bone marrow supplemented with S-adenosylmethionine converts benzene into toluene, which in turn yields o -, m - and p -xylenes [F853]. This may account for the apparent hydroxymethylation of benzene (Sloane, N.H., Biochim. Biophys. Acta (1965), 107, 599; Sloane, N.H., Heinemann, M., Biochim. Biophys. Acta (1970), 201, 384).

2. Polunuclear hydrocarbons

Rat is able to incorporate a methyl group at positions 7 and 12 of benzanthracene and possibly its metabolites. Incorporation was not observed with a range of non-carcinogenic analogues [F534]. The enzyme has been located in rat lung cytosol. Incorporation of a second methyl group occurs with both monomethylated benzanthracenes to yield 7,12-dimethylbenzanthracene, with S-adenosylmethionine as the methyl donor [F742, G491]. With rat liver cytosol chrysene forms 6-methylchrysene [G118].

3. Natural products.

Euglena gracilis g-tocopherol methyltransferase (c.f E.C. 2.1.1.64), molecular weight 150000 and optimum pH 7.5, incorporates a methyl group into both g-tocopherol and b-tocopherol, in both cases forming a-tocopherol [G754].

In Streptomyces antibioticus 3-hydroxyanthranilate 4-C-methyltransferase

(E.C. 2.1.1.97), a key enzyme in the formation of actinomycin, yields 3-hydroxy-4-methylanthranilate. The enzyme, molecular weight 36 000, which is stimulated by EDTA or mercaptoethanol, has negligible activity towards a range of substrate analogues [E725, F934]. The co-substrate is S-adenosylmethionine [E676]. The optimum pH lies between 6 and 8, depending on the buffer [E542].

C-Formylation

Rat brain and liver mitochondria incorporate a formyl group at the 5-position of 2,3,4,5-tetrahydro-1-(1-phenylcyclohexyl)pyridinium, with N5-formyltetrahydrofolate as co-substrate [G209].

Incorporation of carboxyl group into phenols without hydroxyl removal

Phenol is a substrate for this reaction type [G923]; bacteria form p -hydroxybenzoate reversibly from phenol and carbon dioxide (E.C. 4.1.1.61), optimum pH 6.5-7.0. A two-stage mechanism has been suggested [F468].

Desulfococcus forms gentisate from quinol and carbon dioxide [H293].

Incorporation of carboxyl group into aniline

Rhodococcus erythropolis cells incorporate carbon dioxide into aniline (optimum at pH 7-7.5) to form anthranilate [D273]. Fratearia also catalyses the reaction [D595].

Incorporation of carboxyl group into polynuclear hydrocarbons

Incorporation of carboxyl group into polynuclear hydrocarbons

Microorganisms act on naphthalene to form 2-naphthoate with incorporation of carbon dioxide. Phenanthrene is also carboxylated, but the position is unclear [K253].

Incorporation of carboxyl group with hydroxyl removal

Phenol is converted into benzoate by microorganisms; the reaction sequence has not been clarified [F177, G281]. Substituted phenols with an ortho substituent yield m-substituted benzoates, with elimination of a hydroxyl group. Substrates include catechol, as well as phenols ortho substituted with halides, amino or carboxyl groups [G917].

A bacterial consortium acts on phenol to form benzoate, apparently involving para carboxylation as well as a dehydroxylation step [G424].

Bacterium forms benzoate from quinol and bicarbonate; it was postulated that gentisate was an intermediate [H110]. Another study found that the reaction occurs in species that dehydroxylate p -hydroxybenzoate [G167].

Other microorganisms, including those found in pig manure form benzoate from phenol, apparently without p -hydroxybenzoate as an intermediate [F515, H842].

The reverse reaction is described under p -Hydroxy- and 3,4-dihydroxybenzoate decarboxylases (below).

Tyrosine phenol-lyase (E.C. 4.1.99.2)

e.g, l-Serine + phenol i l-tyrosine

Citrobacter freundii enzyme has a broad optimum from pH 6.8 to above 9 and acts on phenol to generate l-tyrosine, with S-(o -nitrophenyl)-l-cysteine as co-substrate [F38]. C. intermedius enzyme forms tyrosine from phenol, glycine and formaldehyde, but only in the presence of an aldolase (E.C. 4.1.2.13) [B693].

Erwinia herbicola enzyme has an optimum pH of about 8 for amino acid synthesis, and is denatured by high concentrations of phenol or catechol [A851]. The enzyme also acts on l-dopa, and the reverse reaction has been observed with phenol, catechol, resorcinol and pyrogallol. In intact cells d-tyrosine is converted into l-tyrosine, presumably by side-chain removal and reconstruction [A708].

Escherichia intermedia enzyme acts on resorcinol to form 2,4-dihydroxy-l-phenylalanine, with S-methyl-l-cysteine as co-substrate, which can be replaced by l-tyrosine; presumably the enzyme releases l-serine from l-tyrosine, which then condenses with resorcinol. The configuration with both d- and l-tyrosine is retained at C-3 [A2242, E401]. Phenols that are substrates include phenol, o- and m -cresol, o-and m -chlorophenol, catechol, resorcinol, pyrogallol and hydroxyquinol [B292]. The enzyme requires pyridoxal phosphate as cofactor; its N-oxide and 2'-hydroxypyridoxal phosphate are not so good as cofactors [A155].

Leptoglossus phyllopus enzyme, which is found in the ventral abdominal gland interconverts phenol and l-tyrosine [A2260].

Symbiobacterium thermophilum enzyme, optimum pH 7 and pI 4.8 is heat stable. It is activated by K+ and NH+, but not by Na+ or Mg2 + , with both d- and l-tyrosine as substrates. It converts catechol into l-dopa, with pyruvate and ammonia as co-substrates [G603].

Activity has been found in Pseudomonas, Xanthomonas, Alcaligenes, Achromobacter, Escherichia, Aerobacter, Erwinia, Proteus, Salmonella and Bacillus, but generally only in a small proportion of the strains tested. In this study it was not found in numerous other bacterial genera, fungi, Actinomycetes or yeasts [A692].

Catechol is a substrate for enzyme found in Erwinia, Symbiobacterium and Citrobacter [e.g. E959, G603, J512], and leads to a potentially viable method for the commercial production of l-dopa.

Indole + l-serine 0l-tryptophan

Enzymes from Daucus carota and Nicotiana tabacum utilize as additional substrates 4-, 5- and 6-fluoroindole, 5-hydroxy-, 5-methoxy- and 5-methylindole. Several other substituted indoles are at best poor substrates [B874].

In Juglans regia the enzyme is associated with a particulate fraction that is not mitochondrial, with optimum pH 7-8 [A818].

The reaction has also been studied in Zea mays [H126].

In Enterobacteriaceae genera the activity (using pyruvate and ammonia instead of serine) has been found only in some strains and species of Escherichia, Kluyvera, Enterobacter, Erwinia and Proteus. Other genera tested and found inactive include Pseudomonas, Azotobacter, Aeromonas, Rhizobium, Alcaligenes, Salmonella and Clostridium. Between four and 475 strains of each genus were tested [A781].

Vibrio enzyme requires pyridoxal phosphate, and can use S-methylcysteine in place of cysteine, optimum pH 8.0 [B85].

E. coli enzyme, optimum pH 9, yields 5-hydroxytryptophan from 5-hydroxyindole [A430].

Salmonella typhimurium enzyme, which requires pyridoxal phosphate for activity, is a complex of two pairs of different subunits [K275]. This and E. coli enzymes catalyze the reverse reaction, but only slowly [E101].

The reverse reaction has been described under Tryptophanase.

L-Tryptophan 2-C-methyltransferase

Streptomyces laurentii enzyme, optimum pH 7.8 (sharp) transfers the methyl group from l-methionine to the nucleus of l-tryptophan to form l-2-methyltryptophan with retention of configuration. Indole-3-pyruvate and d-tryptophan (poor) are also substrates, but not indole [K752].

L-Tryptophan 4-dimethylallyltransferase

This compound, which is a key intermediate in the formation of ergot alkaloids, is formed from l-tryptophan in Claviceps paspali [A3599].

Aspulvinone dimethylallyltransferase

Aspergillus terreus enzyme, molecular weight 240 000-270 000 (monomer 45 000) and optimum pH 7.0 acts on aspulvinone E or aspulvinone G and dimethylallyl pyrophosphate to form a series of dimethylallyl-substituted analogues [K829].

Trihydroxypterocarpan dimethylallyltransferase

Glycine enzyme acts on 3,6a,9-trihydroxyptero-carpan and dimethylallyl pyrophosphate to form 2- and 4-dimethylallyl-3,6a,9-trihydroxypterocar-pan [K905]. The enzyme is found after elicitor treatment (Phytophthora megasperma) [K904].

Formation of ubiquinone, tocopherol and analogues by prenylation

In man 2,3-dimethoxy-5-methyl-^ -quinone (ubiquinone 0) is precursor to ubiquinones 30, 45 and 50, and 'menadione diphosphate' is precursor for vitamins K2(20), K2(45) and K2(50) [A774].

Rat liver and brain mitochondria contain 4-hydroxybenzoate: polyprenyl transferase (c.f. E.C. 2.5.1.31), with polyprenyl pyrophosphates containing 8 /10 isoprene units as the second substrate. p -Aminobenzoate is also a substrate. Inhibitors (not very effective) include serotonin, dopamine, noradrenaline, aspirin and other salicylates. Maximum activity is found in heart, and it is also found in kidney and spleen [A147].

Homogentisate is the substrate from which many prenylated compounds are formed.

Benzaldehyde side chain carboligation

3-Octa- and 3-nonaprenyltoluquinols are formed in Euglena gracilis and sugar beet, with octaprenyl- and nonaprenyl pyrophosphates as co-substrates respectively; g-tocopherol is another product [A201, A1584, G754]. Plastoquinones, a- and g-tocopherol are formed in spinach and lettuce chloroplasts, with phytyltoluquinone as a possible intermediate [A2522, D902].

Saccharomyces (baker's yeast) contains a mitochondrial 4-hydroxybenzoate: polyprenyl transferase with an optimum at pH 7 that synthesizes the corresponding 2-polyprenylphenols with two, three, four, five, eight, nine and 10 isoprene units. Formation of the triprenyl product requires trans, trans - farnesyl pyrophosphate. The synthesis is stimulated by Mg2+ and inhibited by phosphate [A147].

An E. coli enzyme system acts on p -hydroxybenzoate and polyprenylpyrophosphates to form 3-octaprenyl-and 3-nonaprenyl-4-hydroxybenzoates [A1524].

Benzaldehyde side chain carboligation

Pseudomonas putida benzoylformate decarboxy-lase (which requires thiamine pyrophosphate) exhibits a second reaction in which benzaldehydes form (R )-benzoins [K424].

respectively [H576].

a. p-Hydroxybenzoate decarboxylase

A microorganism enzyme, molecular weight 420000 (subunit molecular weight 119 000) and pI 5.6 acts on p -hydroxybenzoate to yield phenol [K573].

A bacterial enzyme, optimum pH 6.5 /7.0 reversibly decarboxylates p -hydroxybenzoate specifically; it requires Mn2+ and phosphate, and is rapidly inactivated by oxygen [F468].

A mixture of microorganisms decarboxylates several benzoates reversibly [G632].

b. 3,4-Dihydroxybenzoate decarboxylase

Clostridium hydroxybenzoicum enzyme, molecular weight 270000 and optimum pH 7.0 appears to be a homotetramer, and is absolutely specific for protocatechuate. The reaction is reversible, in favour of the acid. No coenzyme is required. A second enzyme was separated in the same study; the N-terminal sequences of these enzymes were different [J184].

6-Methylsalicylate decarboxylase (E C. 4.1.1.24)

This activity, which forms m -cresol is found in Valsa friesii [A1049].

Orsellinate decarboxylase (E C. 4.1.1.58)

4.2 Decarboxylation reactions of phenolic groups without hydroxylation p -Hydroxy- and 3,4-dihydroxybenzoate decarboxylases (E.C. 4.1.1.61 and E.C. 4.1.1.63 respectively)

Clostridium hydroxybenzoicum enzyme, a homohexamer, subunit molecular weight 57 000, pI 5.1 and optimum pH 5.6-6.2, reversibly decarboxylates p -hydroxybenzoate and protocatechuate to form phenol and catechol

Gliocladium roseum enzyme catalyzes the formation of orcinol and carbon dioxide; the enzyme is activated by azide [K908].

Pyrocatechuate decarboxylase (E.C. 4.1.1.46)

Pyrocatechuate 0 catechol + CO2

An enzyme in Aspergillus niger is a homotetra-mer, monomeric molecular weight 28 000 and optimum pH 5.2 [E417]. Another study found the native molecular weight to be 150000, but with the same optimum pH. It is inhibited by cyanide and borohydride [B83]. A. oryzae enzyme, which does not require cofactors, appears to have a histidyl residue at the active centre [J206].

Bacterium decarboxylates pyrocatechuate as well as gallate, protocatechuate and m-hydroxybenzoate [C396].

Trichosporon cutaneum is a dimer, molecular weight 66000. Other substrates include 2,3,5- and 2,3,6-trihydroxybenzoates [B790].

Gentisate decarboxylase (E.C. 4.1.1.62)

Klebsiella aerogenes enzyme, optimum pH 5.9, which is soluble, does not require oxygen and releases carbon dioxide [K943].

Gallate decarboxylase (E.C. 4.1.1.59)

Pantoea (formerly Enterobacter) agglomerans enzyme, a homohexamer, molecular weight 320 000, requires Fe (which makes it unique among similar decarboxylases), and is inhibited by Fe2 +-binding reagents. It is highly specific, forming pyrogallol and carbon dioxide [J859].

A bacterium has been described that decarboxylates gallate and several other benzoates [C396].

3.4-Dihydroxyphthalate decarboxylase

Protocatechuate is formed from 3,4-dihydroxyphthalate in Micrococcus; no enzymology has been described [K753].

4.5-Dihydroxyphthalate carboxylyase

4,5-Dihydroxyphthalate 0 protocatechuate + CO2

Pseudomonas fluorescens enzyme is probably a hexamer, molecular weight 420 000, monomeric molecular weight 66 000 and optimum pH 6.8. It is inhibited by p -chloromercuribenzoate [D538]. P. testosteroni enzyme, which is induced by phthalate, appears to be a tetramer, molecular weight 150 000 and monomeric molecular weight 38 000. It also forms m-hydroxybenzoate from 4-hydroxyphthalate [A3332].

p-Cresol formation from p-hydroxyphenylacetate

Clostridium difficile enzyme, which is unstable, requires amino acids (serine or threonine) or the corresponding pyruvates as cofactor, or dithionite [D917].

4.3 Decarboxylation reactions of side-chains

Auxin oxidases

Zea mays enzyme is composed of two isozymes, molecular weights 32 500 (main) and 54 500, with a requirement for Mn2+ and p -coumarate. The product with IAA is indole-3-methanol [F798]. This product is also found in wheat [D150] and Pinus sylvestris [D597]. Lupinus alba forms, in addition, 3-hydroxymethyloxindole, 3-methyleneoxindole and 3,3'-bisindolylmethane [F847]. Horseradish peroxidase also forms indole-3-methanol, 3-formylindole, 3-methyleneoxindole and 3-hydroxymethyloxindole from IAA [C525, D234, F961].

3-Methyleneoxindole formation (indoleacetate oxidase)

Indole-3-acetate 0 3-methyleneoxindole

Arachis contains four peroxidase isozymes with indoleacetate oxidase and polyphenol oxidase activities. Each isozyme has a different optimum pH for each type of substrate; the optima for indoleacetate oxidase are 7.2-7.6 [A2519].

Bean (Phaseolus vulgaris) etiolated seedling root oxidase activity is increased by treating the plants with naphthenate (identity unspecified), cyclohexanecarboxylate and cyclopentylacetate; there is no in vitro effect with these compounds [A220].

Three commercial sources of horseradish peroxidase contain a total of 42 isozymes by isoelectric focussing, and the oxidase-peroxidase ratio is essentially identical for all these isozymes [A3293]. Other studies have failed to separate peroxidase and indole oxidase activities in peroxidase preparations from horseradish and Betula (yellow birch) leaves (20 and 13 isozymes respectively) [A3428, A3429].

Studies on Nicotiana indicate that the reaction is entirely mediated by peroxidases. At least four isozymes are found by electrophoresis, and indoleacetate oxidase is separated from monophenol monooxygenases [A221].

Oat coleoptile peroxidase is separable by electrophoresis into eight isozymes, six of which exhibit indole-3-acetate oxidase activity [A1135].

Peach seed enzyme, optimum pH 4.5-5.0, requires Mn2+ and 2,4-dichlorophenol [A655].

Wheat enzyme is a peroxidase, which requires Mn2+ and a phenolic cofactor without addition of peroxide. With peroxide, ferulate and p -coumarate are also oxidized. A coloured free radical appears to be the first product [A219].

4-Hydroxymandelate oxidase (E C. 1.1.3.19)

Pseudomonas convexa enzyme, molecular weight 155 000 requires Mn2 +, oxygen and FAD or FMN in oxidatively decarboxylating 4-hydroxy-mandelate to form p -hydroxybenzaldehyde. It is inhibited by thiols, EDTA, cyanide and 8-hydroxyquinoline [A2681].

quinone methide, with oxidative decarboxylation [D865, E963].

Benzoylformate (phenylglyoxylate) decarboxylase

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