A para-site-specific hydroxylation of various aromatic compounds by Mycobacterium sp. strain 12523

A screening of microorganisms with para-site-specific hydroxylation activity of aromatic compounds has been carried out by a three-step screening procedure involving a newly established micro-plate assay method. About 1300 strains isolated from about 5000 soils showed the activity. The hydroxylation of aromatic compounds by one of the isolates, designated Mycobacterium sp. strain 12523, which had the highest activity, was examined in detail. It produced p-phenols with high selectivity without o-or m-site hydroxylation. The yield of hydroquinone from phenol by strain 12523 was 97 mol%.     

Assimilation of Propane and Characterization of Propane Monooxygenase from Rhodococcus erythropolis3/89

The ability of propane-assimilating microorganisms of the genus Rhodococcusto utilize metabolites of the terminal and subterminal pathways of propane oxidation was studied. Propane monooxygenase of Rhodococcus erythropolis3/89 was shown to be an inducible enzyme catalyzing epoxidation and hydroxylation of organic compounds. The optimum conditions for the epoxidation of gaseous and liquid alkenes and the hydroxylation of aromatic carbohydrates were found.     

Conjugative transfer of preferential utilization of aromatic compounds from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CSV86

Pseudomonas putida CSV86 utilizes naphthalene (Nap), salicylate (Sal), benzyl alcohol (Balc), and methylnaphthalene (MN) preferentially over glucose. Methylnaphthalene is metabolized by ring-hydroxylation as well as side-chain hydroxylation pathway. Although the degradation property was found to be stable, the frequency of obtaining Nap⁻Sal⁻MN⁻Balc⁻ phenotype increased to 11% in the presence of curing agents. This property was transferred by conjugation to Stenotrophomonas maltophilia CSV89 with a frequency of 7 × 10⁻⁸ per donor cells. Transconjugants were Nap⁺Sal⁺MN⁺Balc⁺ and metabolized MN by ring- as well as side-chain hydroxylation pathway. Transconjugants also showed the preferential utilization of aromatic compounds over glucose indicating transfer of the preferential degradation property. The transferred properties were lost completely when transconjugants were grown on glucose or 2YT. Attempts to detect and isolate plasmid DNA from CSV86 and transconjugants were unsuccessful. Transfer of degradation genes and its subsequent loss from the transconjugants was confirmed by PCR using primers specific for 1,2-dihydroxynaphthalene dioxygenase and catechol 2,3-dioxygenase (C23O) as well as by DNA–DNA hybridizations using total DNA as template and C23O PCR fragment as a probe. These results indicate the involvement of a probable conjugative element in the: (i) metabolism of aromatic compounds, (ii) ring- and side-chain hydroxylation pathways for MN, and (iii) preferential utilization of aromatics over glucose.     

Studies on plant growth-regulating substances

The metabolism of certain 2,6-disubstituted phenols that possess high auxin activity in the pea segment, pea curvature and tomato-leaf epinasty tests, but are much less active in the wheat cylinder test, has been investigated in wheat, pea and tomato tissue. Metabolites were identified by thin-layer chromatography and a semi-quantitative assay method was developed. The low activity of 2,6-dihalogenophenols and inactivity of 2-halogeno-6-nitro-phenols and 3-halogeno-2-hydroxybenzonitriles in the wheat cylinder test was caused by rapid metabolic conversion of the compounds in this tissue to inactive compounds by a process involving hydroxylation of the aromatic ring in the para- position. No such inactivation occurred in pea and tomato tissues. Evidence for a novel detoxification of nitrophenols within both pea and wheat tissue was obtained; 2-bromo–6-nitrophenol was converted via 2-bromo-6-aminophenol to N-acetyl-2-bromo-6-aminophenol. Certain 3-halogeno-2-hydroxybenzaldehydes and corresponding aceto-phenones, although fulfilling the necessary structural and electronic criteria for auxin activity, are inactive. Metabolic studies indicate that this is because they are metabolized in wheat, pea and tomato tissues to compounds not possessing the structural requirements for auxin activity.     

A para-site-specific hydroxylation of aromatic compounds by Mycobacterium sp. strain 12523: stabilization of the hydroxylation activity

Mycobacterium sp. strain 12523 has a para-site-specific hydroxylation activity, which produce para-substituted phenols from various aromatic compounds. However, the activity is unstable and the reactions are inactivated within 24 h. In order to extend the reaction period, the factors that affected reaction stability were examined. The hydroxylation activity of the cells incubated in buffer was significantly stabilized by the inclusion of an inducer such as methyl ethyl ketone. It is suggested that a regulatory mechanism is involved in controlling the activity. This study resulted in the development of a convenient method to stabilize the hydroxylation activity, involving the addition of an inducer, such as acetone, to the reaction system. This method permitted the hydroxylation reaction to continue for more than 67 h. Received: 27 January 1997 / Received revision: 18 March 1997 / Accepted: 13 April 1997     

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

A model (NADH-phenazine methosulfate-O₂) 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 Fe³⁺ and Cu²⁺ 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 H₂O₂ 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 O₂ to generate O₂ which could be subsequently protonated to form the perhydroxyl radical.     

Nitration of Tyrosine by Hydrogen Peroxide and Nitrite

Peroxynitrite anion is a powerful oxidant which can initiate nitration and hydroxylation of aromatic rings. Peroxynitrite can be formed in several ways, e.g. from the reaction of nitric oxide with superoxide or from hydrogen peroxide and nitrite at acidic pH. We investigated pH dependent nitration and hydroxylation resulting from the reaction of hydrogen peroxide and nitrite to determine if this reaction proceeds at pH values which are known to occur in vivo. Nitration and hydroxylation products of tyrosine and salicylic acid were separated with an HPLC column and measured using ultraviolet and electrochemical detectors. These studies revealed that this reaction favored hydroxylation between pH 2 and pH4, while nitration was predominant between pH 5 and pH 6. Peroxynitrite is presumed to be an intermediate in this reaction as the hydroxylation and nitration profiles of authentic peroxynitrite showed similar pH dependence. These findings indicate that hydrogen peroxide and nitrite interact at hydrogen ion concentrations present under some physiologic conditions. This interaction can initiate nitration and hydroxylation of aromatic molecules such as tyrosine residues and may thereby contribute to the biochemical and toxic effects of the molecules.     

Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from <Emphasis Type="Italic">Sphingobium yanoikuyae</Emphasis> B1

Background  

The initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-F_B1). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-O_B1), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo[a]pyrene.     

Inhibition of Zinc Metallopeptidases by Flavonoids and Related Phenolic Compounds: Structure-Activity Relationships

Abstract

Flavonoids and other benzopyrone substances, having an appropriate hydroxylation profile, may inhibit the metalloenzymes leucine aminopeptidase (LAP), aminopeptidase M (AP-M), and carboxypeptidase A (CP-A). A structural feature that evidently favours the interaction between flavonoids and the three metalloenzymes is the 2,3-double bond conjugating the A and B rings and conferring a planar structure. This can be considered virtually indispensable for inhibition of the three metallopeptidases, though the hydroxylation profile required differed for each of the enzymes, and the interaction mechanism and behaviour also differed. The inhibitory effect of flavonoids on LAP was reversible, and to be effective the flavonoid had to have conjugated A and B rings and or tho-dihydroxylation on at least one of the aromatic rings. This same requirement was essential for inhibition by coumarins and was attributed to a catechol-like mechanism of interaction. The inhibitory effects on AP-M were due to inactivation of the enzyme, irreversibly altered by flavonoids with a 2,3-double bond and a minimum of one hydroxyl substituent on each of the aromatic rings. With CP-A, conjugation of the A and B rings enhanced the inhibitory effect of flavonoids, though it was not strictly required. The interaction between the polyphenolic substances tested and the two zinc aminopeptidases was not reversed by adding zinc to the reaction medium, indicating that the inhibition is not due to the coordination of the phenolic hydroxyl groups with the catalytical zinc of active site, though the presence of zinc affected the interaction behaviour differently according to each substance's hydroxylation profile.     

Hydroxylation of daidzein by CYP107H1 from Bacillus subtilis 168

CYP107H1, from Bacillus subtilis 168 known as fatty acid hydroxylase, showed the ortho-specific hydroxylation activity to daidzein, when coupled to the putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida as the redox partners. The electron transfer system of the three proteins was constructed in Escherichia coli BL21 (DE3) system using the two plasmids containing different selection markers. The daidzein hydroxylation was demonstrated with recombinant whole cell and in vitro system using the artificial redox partner for electron transfer. The identification of the hydroxylation reaction yielding 7,3′,4′-trihydroxyisoflavone was elucidated using gas chromatography mass spectrometry (GC–MS). This oxidizing activity of CYP107H1 towards daidzein represents the new hydroxylation of aromatic compound as substrate.     

CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium

CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and β-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6′β-hydroxy-lovastatin, 3′α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-β-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-β-damascone, and 3,4-epoxy-β-damascone). Besides that, a novel compound, 2-hydroxy-β-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.

     

Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase–peroxygenase

Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ ₃₀₃ = 2,010 M⁻¹ cm⁻¹), and the apparent Michaelis–Menten (K _m) and catalytic (k _cat) constants for the reaction were estimated to be 320 μM and 166 s⁻¹, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase–peroxygenases.     

Side Chain Hydroxylation of Aromatic Compounds by Fungi. Part 6. Biotransformation of Olefins by Mortierella Isabellina

The fungus Mortierella isabellina ATCC 42613 converts phenyl-substituted olefins to vicinal diols. The substrate selectivity and product stereochemistry of this biotransformation can be rationalized by application of the model developed for benzylic hydroxylation of aromatic hydrocarbons by M isabellina.     

On the mechanism of oxygen activation by tetrahydropterin and dihydroflavin-dependent monooxygenases

A brief critical survey of the current theories of molecular oxygen activation in the hydroxylation of aromatic compounds by dihydroflavin- and tetrahydropterin-dependent monooxygenases is presented. A reinterpretation of the available data has resulted in the proposal of a new mechanistic hypothesis. It is suggested that the dihydroflavin (or tetrahydropterin) cofactor interacts with ground-state molecular oxygen to give a C_4a-C_10a singlet perepoxy dihydroflavin (or a C_4a-C_8a perepoxy tetrahydropterin) intermediate which functions as the oxenoid reagent in this class of biological hydroxylations.     

Degradation of 4-nitrophenol by the lignin-degrading basidiomycete <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The fungal metabolism of 4-nitrophenol (4-NP) was investigated using the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Despite its phenolic feature, 4-NP was not oxidized by extracellular ligninolytic peroxidases. However, 4-NP was converted to 1,2-dimethoxy-4-nitrobenzene via intermediate formation of 4-nitroanisole by the fungus only under ligninolytic conditions. The metabolism proceeded via hydroxylation of the aromatic ring and methylation of phenolic hydroxyl groups. Although the involvement of nitroreductase in the metabolism of 2,4-dinitrotoluene by many aerobic and anaerobic microorganisms including P. chrysosporium has been reported, no formation of 4-aminophenol was observed during 4-NP metabolism. The formation of 1,2-dimethoxy-4-nitrobenzene was effectively inhibited by exogenously added piperonyl butoxide, a cytochrome P450 inhibitor, suggesting that cytochrome P450 is involved in the hydroxylation reaction. Thus, P. chrysosporium seems to utilize hydroxylation and methylation reactions to produce a more susceptible structure for an oxidative metabolic system.     

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.     

Regulation of biosynthesis ofN-glycolylneuraminic acid-containing glycoconjugates: characterization of factors required for NADH-dependent cytidine 5′monophosphate-N-acetylneuraminic acid hydroxylation

The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochromeb ₅. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochromeb ₅, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochromeb ₅, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression ofN-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation.Abbreviations CMP cytidine 5-monophosphate - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - DTT dithiothreitol     

Inhibitory Effect of Aryl Thienyl-Ketones and - Thioketones on Arachidonic Acid-induced Malondialdehyde Formation in Human Platelets: Biological Data and Molecular Modelling

Abstract

A series of anti-thrombotic aryl thienyl-ketones and -thioketones was assayed in vitro for their inhibitory effect on malondialdehyde (MDA) production induced by arachidonic acid in human platelets. For several compounds MDA formation was strongly inhibited indicating that the anti-platelet target was situated on the cyclooxygenase pathway. A comparison between the inhibition constant K₁ and the IC₅₀ values revealed competitive inhibition kinetics. The molecular structure of one active compound was analysed by X-ray diffraction and theoretical calculations to provide information on its electronic and lipophilic properties.     

Induction of mitotic crossing over in Saccharomyces by p-Toluidine.

Summary p-Toluidine, a carcinogen for rats, does not cause genetic damage when tested directly in Saccharomyces cerevisiae; however, certain chemical derivatives of p-toluidine do induce gene conversion when tested directly. It may be suspected by analogy with other aromatic amines that p-toluidine, a monocyclic aromatic amine, requires conversion to breakdown products which are then the genetically active and carcinogenic entities. The Udenfriend hydroxylation medium, which has been used previously to show the genetic activity of certain other aromatic amines and nitrosamines, was used in the incubation of p-toluidine with Saccharomyces cerevisiae. The resulting breakdown products, but not the parent compound, induced reciprocal mitotic recombination in a diploid strain D-3. Recombination was monitored by using induced homozygosity of the red ade2 marker, and the reciprocal nature of the event was confirmed by observing the simultaneous homozygosity of two peripheral markers.     

Construction and assessment of reaction models between F1F0-synthase and organotin compounds: molecular docking and quantum calculations

Organotin compounds are the active components of some fungicides, which are potential inhibitors of the F₁F₀-ATP synthase. The studies about the reaction mechanism might indicate a pathway to understand how these compounds work in biological systems, however, has not been clarified so far. In this line, molecular modeling studies and density functional theory calculations were performed in order to understand the molecular behavior of those compounds when they interact with the active site of the enzyme. Our findings indicate that a strong interaction with His132 can favor a chemical reaction with organotin compounds due to π–π stacking interactions with aromatic rings of organotin compounds. Furthermore, dependence on molecule size is related to possibility of reaction with the amino acid residue His132. Thus, it can also be noticed, for organotin compounds, that substituents with four carbons work by blocking the subunit a, in view of the high energy transition found characterized by steric hindrance.