Study of alpha-methyldopa oxidation by tyrosinase

The pathway for alpha-methyldopa oxidation to alpha-methyldopachrome, by mushroom tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometry determination have both been undertaken. The steps for alpha-methyldopa transformation into its aminochrome would be: alpha-methyldopa----o-alpha-methyldopaquinone-H+----o-alpha- methyldopaquinone----leuko-alpha-methyldopachrome----alpha- methyldopachrome. The stoichiometry for this conversion corresponded to the equation: 2 o-alpha-methyldopaquinone-H+----alpha-methyldopa + alpha-methyldopachrome. At very acid pH values, another route implying the addition of water to the quinonic ring, competes with the first one. Two chemical pathways can be proposed from alpha-methyldopaquinone-H+, the relative importance of which is determined by the pH. A theoretical and experimental kinetic approach was applied to this oxidative reaction. Rate constants and thermodynamic activation parameters of the chemical steps, have been evaluated. The results obtained confirmed that alpha-methyldopa oxidation by tyrosinase followed a scheme similar to that established for L-dopa and alpha-methylnoradrenaline.     

油菜素内酯生物合成途径的研究进展

油菜素内酯(BRs)在植物的生长发育过程中具有重要作用。该文首先综述了油菜素甾醇的结构及其生物合成途径的研究方法。之后, 介绍了其化学及生物活性的检测方法。最后, 详细介绍了BR生物合成的早期和晚期C-6氧化途径及早期C-22和C-23羟化与合成途径的调控, 并阐述了近年来植物油菜素内酯生物合成缺失突变体及其合成酶等方面的研究进 展。     

Hydroxylation of biphenyl by the yeast Trichosporon mucoides

Hydroxylation of biphenyl by the dibenzofuran-degrading yeast Trichosporon mucoides SBUG 801 was studied. Glucose-grown cells degraded 40% of the biphenyl added within the first 24 h of incubation. The first step in the biotransformation pathway was the monohydroxylation of the biaryl compound to produce 2-, 3-, and 4-hydroxybiphenyl. Further oxidation produced seven dihydroxylated intermediates; the second hydroxyl group was added either on the aromatic ring already hydroxylated or on the second ring. Of all metabolites, 2,5-dihydroxybiphenyl accumulated in the supernatant in the highest concentration. The initial hydroxylation favors the 4-position to produce 4-hydroxybiphenyl, which is subsequently hydroxylated to form 3,4-dihydroxybiphenyl. When biphenyl was replaced as a substrate by 4-hydroxybiphenyl, further hydroxylation of the intermediate 3,4-dihydroxybiphenyl resulted in 3,4,4'-trihydroxybiphenyl. Incubation of T. mucoides with biphenyl and 18O2 indicated a monooxygenase-catalyzed reaction in the oxidation of biphenyl. The hydroxylation was inhibited by 1-aminobenzotriazole and metyrapone, known cytochrome P450 inhibitors. These results are very similar to those observed in the biotransformation of biphenyl in mammals.     

Biochemistry of lactone formation in yeast and fungi and its utilisation for the production of flavour and fragrance compounds

The consumers’ demand for natural flavour and fragrances rises. To be natural, compounds have to result from the extraction of natural materials and/or to be transformed by natural means such as the use of enzymes or whole cells. Fungi are able to transform some fatty acids into lactones that can thus be natural. Although some parts of this subject have been reviewed several times, the present article proposes to review the different pathways utilised, the metabolic engineering strategies and some current concerns on the reactor application of the transformation including scaling up data. The main enzymatic steps are hydroxylation and β-oxidation in the traditional way, and lactone desaturation or Baeyer–Villiger oxidation. Although the pathway to produce γ-decalactone is rather well known, metabolic engineering strategies may result in significant improvements in the productivity. For the production of other lactones, a key step is the hydroxylation of fatty acids. Beside the biotransformation, increasing the production of the various lactones requires from biotechnologists to solve two main problems which are the toxicity of lactones toward the producing cell and the aeration of the emulsified reactor as the biochemical pathway is very sensitive to the level of available oxygen. The strategies employed to resolve these problems will be presented.     

An electron spin resonance study of o-semiquinones formed during the enzymatic and autoxidation of catechol estrogens

Electron spin resonance spectroscopy has been used to demonstrate production of semiquinone-free radicals from the oxidation of the catechol estrogens 2- and 4-hydroxyestradiol and 2,6- and 4,6-dihydroxyestradiol. Radicals were generated either enzymatically (using horseradish peroxidase-H2O2 or tyrosinase-O2) or by autoxidation, and were detected as their complexes with spin-stabilizing metal ions (Zn2+ and/or Mg2+). In the peroxidase system, radicals are produced by one-electron oxidation of the catechol estrogen and their decay is by a second-order pathway, consistent with their disproportionation to quinone and catechol products. With tyrosinase-O2, radical generation occurs indirectly. Initial hydroxylation of phenolic estrogen (at either the 2- or 4-position) gives a catechol estrogen in situ; subsequent two-electron oxidation of the catechol to the quinone, followed by reverse disproportionation, leads to the formation of radicals. A competing mechanism for radical production involves autoxidation of the catechol. Results obtained from the estrogen systems have been compared with those from the model compound 5,6,7,8-tetrahydro-2-naphthol.     

Effect of pH on the oxidation pathway of dopamine catalyzed by tyrosinase

The oxidation of 3,4-dihydroxyphenylethylamine (dopamine) by O2 catalyzed by tyrosinase yields 4-(2-aminoethyl)-1, 2-benzoquinone (o-dopaminequinone), which evolves nonenzymatically through two branches or sequences of reactions, whose respective operations are determined by the pH of the medium. The cyclization branch of o-dopaminequinone takes place in the entire range of pH and is the only significant branch at pH greater than or equal to 6. The hydroxylation branch of o-dopaminequinone only operates significantly at pH less than 6, and involves the accumulation of 2,4,5-trihydroxyphenylethylamine (6-hydroxydopamine) and 5-(2-aminoethyl)-2-hydroxy-1,4-benzoquinone (p-topaminequinone), identified from cyclic voltammetry assays. The kinetic characterization of the hydroxylation branch of o-dopaminequinone has been carried out by spectrophotometric and oxymetric assays. The successful fitting of data to the kinetic behavior predicted by the kinetic analysis at both pH greater than or equal to 6 and pH less than 6 confirms the overall oxidation pathway proposed for the dopamine oxidation catalyzed by tyrosinase. The antitumoral power of dopamine is possibly enhanced by the high cytotoxicity of 6-hydroxydopamine and p-topaminequinone, accumulated at the acidic pH characteristic of melanosomes and melanome cells.     

Two different primary oxidation mechanisms during biotransformation of thymol by gram-positive bacteria of the genera Nocardia and Mycobacterium

Thymol has antibacterial, antifungal, insecticidal, and antioxidative properties which are the basis for the wide use of this compound in the cosmetic, food, and pharmaceutical industries. Although thymol is a ubiquitously occurring substance in the environment, data about its degradation and detoxification by bacteria are sparse. Here, we show the existence of two different pathways for the biotransformation of thymol by Nocardia cyriacigeorgica and Mycobacterium neoaurum which were described for the first time for gram-positive bacteria. The first pathway starts with hydroxylation of thymol to thymohydroquinone (2-isopropyl-5-methylbenzene-1,4-diol) with subsequent oxidation to thymobenzoquinone (2-isopropyl-5-methyl-1,4-benzoquinone). The second pathway involves hydroxylation of the methyl group followed by oxidation to 3-hydroxy-4-isopropylbenzoic acid, possibly via the aldehyde 3-hydroxy-4-isopropylbenzaldehyde. It is noteworthy that the branched side chain of thymol was not oxidized. Similarities and differences of these oxidation processes with those of the gram-negative bacterium Pseudomonas putida, fungi, and plants are discussed and, in addition, the toxicity of thymol towards N. cyriacigeorgica and M. neoaurum was tested. The experiments showed a temporary growth inhibition with 0.025 % thymol. This was explained by degradation of thymol and the formation of products which are less toxic than thymol itself.     

植物三萜皂苷生物合成中关键后修饰酶研究进展

三萜皂苷是由三萜苷元、糖基、糖醛酸等组成的C30萜类化合物,是许多药用植物的主要活性成分,具有广泛的药理作用。三萜皂苷的生物合成包括前体和三萜皂苷骨架的形成以及调控皂苷结构多样性的后修饰。三萜皂苷的后修饰包括三萜骨架的氧化/羟基化和糖基化,分别由不同超基因家族编码的细胞色素P450单加氧酶和糖基转移酶进行催化。三萜皂苷通过后修饰最终可形成多种单体皂苷。目前,已在少数植物中识别和确认了个别与三萜皂苷生物合成相关的关键后修饰酶,发现了部分很可能参与后修饰过程的候选基因。该文就近年来国内外有关三萜皂苷生物合成途径关键后修饰酶的研究进行综述,为进一步开展相关研究和对合成精细途径的解析提供参考。     

Hydroxylation of the mycotoxin zearalenone at aliphatic positions: novel mammalian metabolites

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species and frequently found as a contaminant of food and feed. Earlier studies have disclosed that ZEN is biotransformed in microsomes from human and rat liver to multiple hydroxylated metabolites, two of which have recently been identified as products of aromatic hydroxylation. Here, we report for the first time on the structure elucidation of metabolites arising through hydroxylation of the aliphatic ring of ZEN at various positions. By using reference compounds and ZEN labeled with deuterium at specific positions, evidence was provided for the preferential hydroxylation of ZEN at C-8 and, to a lesser extent, at C-9, C-10, and C-5. In contrast, hydroxylation at C-6 could be ruled out, as could oxidation of the olefinic double bond. These results imply that the phase I metabolism of ZEN in the mammalian organism is more extensive than previously thought, and warrant further studies on the in vivo formation of the novel ZEN metabolites and their biological activities.     

Oxygenation of Hexadecane in the Biosynthesis of Cyclic Glycolipids in Torulopsis Apicola

Biotransformation of [1-¹³C] labelled hexadecane, hexadecanol and hexadecanoic acid have been investigated using the yeast Torulopsis apicola. The yeast produces a microcrystalline mixture of two glycolipids, the lipophilic moiety of which consists of ω- or (ω-l)-hydroxylated hexadecanoic acid. Biosynthesis of these glycolipids takes place via hydroxylation of hexadecane, oxidation to hexadecanoic acid and ω or (ω-l)-hydroxylation of hexadecanoic acid. Feeding the cell cultures with a mixture of hexadecane and [1-¹³C] labelled hexadecane derivatives one observes ¹³C enrichment ratios which indicate that neither of the biohydroxylation or oxidation steps are rate limiting in the formation of the glycolipids, furthermore, two different monooxygenase systems appear to be involved in hydroxylation of hexadecane and hexadecanoic acid.     

A kinetic analysis of Drosophila melanogaster dopa decarboxylase

The kinetic mechanism of dopa decarboxylase (3,4-dihydroxy-L-phenylalanine carboxy-lyase, EC 4.1.1.28) was investigated in Drosophila melanogaster. Based on initial velocity and product inhibition studies, an ordered reaction is proposed for dopa decarboxylase. This kinetic mechanism is interpreted in the context of measured enzyme activities and the catecholamine pools in Drosophila. The 1(2)amd gene is immediately adjacent to the gene coding for dopa decarboxylase (Ddc) and determines hypersensitivity to alpha-methyldopa in Drosophila. Dopa decarboxylase does not decarboxylate alpha-methyldopa and hence does not generate a toxic product capable of inhibiting 1(2)amd gene function. We propose that the 1(2)amd gene is involved with an unknown catecholamine pathway involving dopa but not dopamine.     

The biosynthesis of monolignols: a "metabolic grid", or independent pathways to guaiacyl and syringyl units?

Lignin is a complex polymer formed by the oxidative polymerization of hydroxycinnamyl alcohol derivatives termed monolignols. The major monolignols in dicotyledonous angiosperm lignin are monomethylated guaiacyl (G) units derived from coniferyl alcohol, and dimethylated syringyl (S) units derived from sinapyl alcohol. The biochemical pathways leading to the formation of monolignols feature successive hydroxylation and O-methylation of the aromatic ring and conversion of the side chain carboxyl to an alcohol function. The current view of the monolignol biosynthetic pathway envisages a metabolic grid leading to G and S units, through which the successive hydroxylation and O-methylation reactions may occur at different levels of side chain oxidation. The present article assesses biochemical and genetic evidence for and against such a model, including recent data on the methylation reactions of monolignol biosynthesis in alfalfa. We draw attention to portions of the currently accepted monolignol pathway that may require revision, and suggest an alternative model in which metabolic channeling allows for independent pathways to G and S lignin.     

A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin

We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases.     

The sexually differentiated metabolism of [6,7-3H]17 beta-oestradiol in rats: male-specific 15 alpha- and male-selective 16 alpha-hydroxylation and female-selective catechol formation.

The oxygenated-metabolite profiles of exogenous 17 beta-oestradiol (E2) in adult male and female Wistar rats have been characterized and major sex-dependent biotransformations observed which correlate with the regioselectivities of known sexually differentiated hepatic P450. [6,7-3H]E2 (27 micrograms/kg) was given i.v. The metabolites of E2 were rapidly and extensively excreted in bile (46 and 78% of the dose over 1 and 6 h, respectively). Female rats metabolized E2 by one major pathway: oxidation to oestrone (E1) followed by C-2 hydroxylation and O-methylation; the principal aglycones (0-1 h bile collections) were E1 (14%), 2-hydroxyE1 (2-OHE1) (42%) and 2-methoxyE1 (24%). Male rats metabolized E2 principally by two parallel composite pathways of E1 hydroxylation which yielded a complex mixture of mono- and di-oxygenated compounds: 15 alpha-OHE1 (33%), 2,15 alpha-diOHE1 (7%), and 2-methoxy-15 alpha OHE1 (14%); 16 alpha-OHE1 (13%), 2,16 alpha-diOHE1 (4%) and 2-methoxy-16 alpha-OHE1 (2%). 15 alpha-Hydroxylation was unique to males. The balance of aromatic and alkyl hydroxylation in males was dose-dependent: at 3 mg/kg, 15 alpha-hydroxylation was decreased approx. 50% in favour of 2-hydroxylation whilst 16 alpha-hydroxylation was largely unaffected. The male-specific 15 alpha-hydroxylation and male-predominant 16 alpha-hydroxylation of E1 derived from E2 in vivo may be ascribable to the male-specific isoforms P450IIC13 and P450IIC11, respectively.     

Aldehyde oxidase 1 gene is regulated by Nrf2 pathway

Aldehyde oxidase is a member of the molybd-flavo enzyme family that catalyzes the hydroxylation of heterocycles and the oxidation of aldehydes into corresponding carboxylic acids. Aldehyde oxidase-1 (AOX1) is highly expressed in liver and is involved in the oxidation of a variety of aldehydes and nitrogenous heterocyclic compounds, including anti-cancer and immunosuppressive drugs. However, the physiological substrates of AOX1 have not been identified, and it was unknown how the expression of AOX1 is regulated. Here, we found that the AOX1 gene is regulated by the Nrf2 pathway. Two Nrf2 binding consensus elements (antioxidant responsive element, ARE) are located in the 5' upstream region of the rat AOX1 gene. Molecular analyses using reporter transfection analysis, EMSA, and ChIP analysis show that Nrf2 binds to and strongly activates the rat AOX1 gene.     

The metabolism of 3,5-di-tert.-butylphenyl N-methylcarbamate in insects and by mouse liver enzymes

The oxidation of 3,5-di-tert.-butylphenyl N-methylcarbamate (Butacarb) has been studied in the flies Musca domestica and Lucilia sericata, grass grubs Costelytra zealandica and the mouse. In all species eleven oxidation products, which were formed by hydroxylation of the tert.-butyl groups and the N-methyl group, were detected.     

Decarboxylation-dependent transamination catalyzed by mammalian 3,4-dihydroxyphenylalanine decarboxylase

In addition to the usual decarboxylation, pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase catalyzes a decarboxylation-dependent transamination which converts dopa into 3,4-dihydroxyphenylacetaldehyde and sinultaneously converts enzyme-bound pyridoxal-P into pyridoxamine-P. Similar reactions occur when this enzyme acts on m-tyrosine, alpha-methyldopa, and alpha-methyl-m-tyrosine. The transamination occurs in about 0.02% of decarboxylations of dopa and m-tyrosine and in about 2% of decarboxylations of alpha-methyldopa and alpha-methyl-m-tyrosine. The fraction of decarboxylations proceeding by the transamination pathway is independent of pH. This reaction appears to result from a divergence in the normal mechanism of decarboxylation; the quinoid intermediate which is formed by decarboxylation of the substrate-pyridoxal-P-Schiff base ordinarily protonates on the alpha carbon of the amino acid, but protonation occasionally occurs at the benzylic carbon of the coenzyme, and this latter route leads to transamination.     

Transformation of some 3α-substituted steroids by Aspergillus tamarii KITA reveals stereochemical restriction of steroid binding orientation in the minor hydroxylation pathway

Aspergillus tamarii contains an endogenous lactonization pathway which can transform progesterone to testololactone in high yield through a sequential four step enzymatic pathway. In this pathway testosterone is formed which primarily undergoes oxidation of the C-17β-alcohol to a C-17 ketone but, can also enter a minor hydroxylation pathway where 11β-hydroxytestosterone is produced. It was recently demonstrated that this hydroxylase could monohydroxylate 3β-hydroxy substituted saturated steroidal lactones in all four possible binding orientations (normal, reverse, inverted normal, inverted reverse) on rings B and C of the steroid nucleus. It was therefore of interest to determine the fate of a series of 3α-substituted steroidal analogues to determine stereochemical effect on transformation. Hydroxylation on the central rings was found to be restricted to the 11β-position (normal binding), indicating that the 3α-stereochemistry removes freedom of binding orientation within the hydroxylase. The only other hydroxylation observed was at the 1β-position. Interestingly the presence of this functional group did not prevent lactonization of the C-17 ketone. In contrast the presence of the 11β-hydroxyl completely inhibited Baeyer–Villiger oxidation, a result which again demonstrates that single functional groups can exert significant control over metabolic handling of steroids in this organism. This may also explain why lactonization of 11β-hydroxytestosterone does not occur. Lactonization of the C-17 ketone was not significantly affected by the 3α-alcohol with significant yields achieved (53%). Interestingly a time course experiment demonstrated that the presence of the 3α-acetate inhibited the Baeyer–Villiger monooxygenase with its activity being observed 24 h later than non-acetate containing analogues. Apart from oxidative transformations observed a minor reductive pathway was revealed with the C-17 ketone being reduced to a C-17β-alcohol for the first time in this organism.