Showing papers on "Monooxygenase published in 2007"

Enzymatic hydroxylation of aromatic compounds

Abstract: Selective hydroxylation of aromatic compounds is among the most challenging chemical reactions in synthetic chemistry and has gained steadily increasing attention during recent years, particularly because of the use of hydroxylated aromatics as precursors for pharmaceuticals. Biocatalytic oxygen transfer by isolated enzymes or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. This review gives an overview of the different enzymes and mechanisms used to introduce oxygen atoms into aromatic molecules using either dioxygen (O2) or hydrogen peroxide (H2O2) as oxygen donors or indirect pathways via free radical intermediates. In this context, the article deals with Rieske-type and α-keto acid-dependent dioxygenases, as well as different non-heme monooxygenases (di-iron, pterin, and flavin enzymes), tyrosinase, laccase, and hydroxyl radical generating systems. The main emphasis is on the heme-containing enzymes, cytochrome P450 monooxygenases and peroxidases, including novel extracellular heme-thiolate haloperoxidases (peroxygenases), which are functional hybrids of both types of heme-biocatalysts.

A biosynthetic gene cluster for a secreted cellobiose lipid with antifungal activity from Ustilago maydis.

Abstract: Summary The phytopathogenic basidiomycetous fungus Ustilago maydis secretes large amounts of the glycolipid biosurfactant ustilagic acid (UA). UA consists of 15,16-dihydroxypalmitic or 2,15,16-trihydroxypalmitic acid, which is O-glycosidically linked to cellobiose at its terminal hydroxyl group. In addition, the cellobiose moiety is acetylated and acylated with a short-chain hydroxy fatty acid. We have identified a 58 kb spanning gene cluster that contains 12 open reading frames coding for most, if not all, enzymes needed for UA biosynthesis. Using a combination of genetic and mass spectrometric analysis we were able to assign functional roles to three of the proteins encoded by the gene cluster. This allowed us to propose a biosynthesis route for UA. The Ahd1 protein belongs to the family of non-haem diiron reductases and is required for α-hydroxylation of palmitic acid. Two P450 monooxygenases, Cyp1 and Cyp2, catalyse terminal and subterminal hydroxylation of palmitic acid. We could demonstrate that infection of tomato leaves by the plant pathogenic fungus Botrytis cinerea is prevented by co-inoculation with wild-type U. maydis sporidia. U. maydis mutants defective in UA biosynthesis were unable to inhibit B. cinerea infection indicating that UA secretion is critical for antagonistic activity.

Substrate Trafficking and Dioxygen Activation in Bacterial Multicomponent Monooxygenases

Abstract: Non-heme carboxylate-bridged diiron centers in the hydroxylase components of the bacterial multicomponent monooxygenases process four substrates during catalysis: electrons, protons, dioxygen, and hydrocarbons. Understanding how protein–protein interactions mediate the transport of these substrates to the diiron center to achieve the selective oxidation of the hydrocarbon is a significant challenge. In this Account, we summarize our current knowledge of these processes with a focus on the methane monooxygenase system. We also describe recent results for the toluene/o-xylene monooxygenase and phenol hydroxylase systems from Pseudomonas sporium OX1. The observation in these latter systems of a diiron(III) oxygenated intermediate having different Mossbauer parameters from analogous species in other carboxylate-bridged diiron proteins is discussed. The results indicate that the ability of the protein framework to tune the reactivity of the diiron center at structurally similar active sites is substantially mo...

Human Cytochrome P450 Family 4 Enzymes: Function, Genetic Variation and Regulation

Abstract: The microsomal cytochrome P450 (CYP) family 4 monooxygenases are the major fatty acid ω-hydroxylases. These enzymes remove excess free fatty acids to prevent lipotoxicity, catabolize leukotrienes and prostanoids, and also produce bioactive metabolites from arachidonic acid ω-hydroxylation. In addition to endogenous substrates, recent evidence indicates that CYP4 monooxygenases can also metabolize xenobiotics, including therapeutic drugs. This review focuses on human CYP4 enzymes and updates current knowledge concerning catalytic activity profiles, genetic variation and regulation of expression. Comparative differences between the human and rodent CYP4 enzymes regarding catalytic function and conditional expression are also discussed.

Arachidonic acid metabolism as a potential mediator of cardiac fibrosis associated with inflammation

Abstract: An increase in left ventricular collagen (cardiac fibrosis) is a detrimental process that adversely affects heart function. Strong evidence implicates the infiltration of inflammatory cells as a critical part of the process resulting in cardiac fibrosis. Inflammatory cells are capable of releasing arachidonic acid, which may be further metabolized by cyclooxygenase, lipoxygenase, and cytochrome P450 monooxygenase enzymes to biologically active products, including PGs, leukotrienes, epoxyeicosatrienoic acids, and hydroxyeicosatetraenoic acids. Some of these products have profibrotic properties and may represent a pathway by which inflammatory cells initiate and mediate the development of cardiac fibrosis. In this study, we critically review the current literature on the potential link between this pathway and cardiac fibrosis.

Cloning of a gene cluster involved in the catabolism of p-nitrophenol by Arthrobacter sp. Strain JS443 and characterization of the p-nitrophenol monooxygenase

Abstract: The npd gene cluster, which encodes the enzymes of a p-nitrophenol catabolic pathway from Arthrobacter sp. strain JS443, was cloned and sequenced. Three genes, npdB, npdA1, and npdA2, were independently expressed in Escherichia coli in order to confirm the identities of their gene products. NpdA2 is a p-nitrophenol monooxygenase belonging to the two-component flavin-diffusible monooxygenase family of reduced flavin-dependent monooxygenases. NpdA1 is an NADH-dependent flavin reductase, and NpdB is a hydroxyquinol 1,2-dioxygenase. The npd gene cluster also includes a putative maleylacetate reductase gene, npdC. In an in vitro assay containing NpdA2, an E. coli lysate transforms p-nitrophenol stoichiometrically to hydroquinone and hydroxyquinol. It was concluded that the p-nitrophenol catabolic pathway in JS443 most likely begins with a two-step transformation of p-nitrophenol to hydroxy-1,4-benzoquinone, catalyzed by NpdA2. Hydroxy-1,4-benzoquinone is reduced to hydroxyquinol, which is degraded through the hydroxyquinol ortho cleavage pathway. The hydroquinone detected in vitro is a dead-end product most likely resulting from chemical or enzymatic reduction of the hypothetical intermediate 1,4-benzoquinone. NpdA2 hydroxylates a broad range of chloro- and nitro-substituted phenols, resorcinols, and catechols. Only p-nitro- or p-chloro-substituted phenols are hydroxylated twice. Other substrates are hydroxylated once, always at a position para to a hydroxyl group.

Altering the substrate specificity and enantioselectivity of phenylacetone monooxygenase by structure-inspired enzyme redesign

Abstract: Of all presently available Baeyer-Villiger monooxygenases, phenylacetone monooxygenase (PAMO) is the only representative for which a structure has been determined. While it is an attractive biocatalyst because of its thermostability, it is only active with a limited number of substrates. By means of a comparison of the PAMO structure and a modeled structure of the sequence-related cyclopentanone monooxygenase, several active-site residues were selected for a mutagenesis study in order to alter the substrate specificity. The M446G PAMO mutant was found to be active with a number of aromatic ketones, amines and sulfides for which wild-type PAMO shows no activity. An interesting finding was that the mutant is able to convert indole into indigo blue: a reaction that has never been reported before for a Baeyer-Villiger monooxygenase. In addition to an altered substrate specificity, the enantioselectivity towards several sulfides was dramatically improved. This newly designed Baeyer-Villiger monooxygenase extends the scope of oxidation reactions feasible with these atypical monooxygenases.

Cloning, expression and characterization of a Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440.

Abstract: The gene encoding a Baeyer-Villiger monooxygenase and identified in Pseudomonas putida KT2440 was cloned and functionally expressed in Escherichia coli. The highest yield of soluble protein could be achieved by co-expression of molecular chaperones. In order to determine the substrate specificity, biocatalyses were performed using crude cell extract, growing and resting cells. Examination of aromatic, cyclic and aliphatic ketones revealed a high specificity towards short-chain aliphatic ketones. Interestingly, some open-chain ketones were converted to the alkylacetates, while for others formation of the ester products with oxygen on the other side of the keto group could also be detected yielding the corresponding methyl or ethyl esters.

Dietary trans 10, cis 12-conjugated linoleic acid reduces the expression of fatty acid oxidation and drug detoxification enzymes in mouse liver.

Abstract: Mice fed diets containing trans 10, cis 12 (t10, c12)-conjugated linoleic acid (CLA) develop fatty livers and the role of the hepatic fatty acid oxidation enzymes in this development is not well defined. We examined the effects of dietary cis 9, trans 11-CLA (c9, t11-CLA) and t10, c12-CLA on the expression of hepatic genes for fatty acid metabolism. Female mice, 8 weeks old, (six animals per group) were fed either a control diet or diets supplemented with 0.5% c9, t11- or c12-CLA for 8 weeks. DNA microarray analysis showed that t10, c12-CLA increased the expression of 278 hepatic genes and decreased those of 121 genes (>2 fold); c9, t11-CLA increased expression of twenty-two genes and decreased those of nine. Real-time PCR confirmed that t10, c12-CLA reduced by the expression of fatty acid oxidation genes including flavin monooxygenase (FMO)-3 95%, cytochrome P450 (cyt p450) 69%, carnitine palmitoyl transferase 1a 77%, acetyl CoA oxidase (ACOX) 50% and PPARalpha 65%: it increased the expression of fatty acid synthase by 3.5-fold (P<0.05 for all genes, except ACOX P=0.08). It also reduced the enzymatic activity of hepatic microsomal FMO by 40% and the FMO3 specific protein by 67%. c9, t11-CLA reduced FMO3 and cyt P450 expression by 61% (P=0.001) and 38% (P=0.06) and increased steoryl CoA desaturase transcription by 5.9-fold (P=0.07). Both decreased fatty acid oxidation and increased fatty acid synthesis seem to contribute to the CLA-induced fatty liver. Since FMO and cyt P450 are also involved in drug detoxification, suppression of the transcription of these genes by CLA may have other health consequences besides development of fatty liver.

tRNA-modifying MiaE protein from Salmonella typhimurium is a nonheme diiron monooxygenase.

Abstract: MiaE catalyzes the posttranscriptional allylic hydroxylation of 2-methylthio-N-6-isopentenyl adenosine in tRNAs. The Salmonella typhimurium enzyme was heterologously expressed in Escherichia coli. The purified enzyme is a monomer with two iron atoms and displays activity in in vitro assays. The type and properties of the iron center were investigated by using a combination of UV-visible absorption, EPR, HYSCORE, and Mossbauer spectroscopies which demonstrated that the MiaE enzyme contains a nonheme dinuclear iron cluster, similar to that found in the hydroxylase component of methane monooxygenase. This is the first example of an enzyme from this important class of diiron monooxygenases to be involved in the hydroxylation of a biological macromolecule and the second example of a redox metalloenzyme participating in tRNA modification.

Construction of a thermostable cytochrome P450 chimera derived from self-sufficient mesophilic parents

Abstract: The P450 monooxygenases CYP102A1 from Bacillus megaterium and CYP102A3 from Bacillus subtilis are fusion flavocytochromes comprising of a P450 heme domain and a FAD/FMN reductase domain. This protein organization is responsible for the extraordinary catalytic activities making both monooxygenases promising enzymes for biocatalysis. CYP102A1 and CYP102A3 are fatty acid hydroxylases that share 65% identity, and their mutants are able to oxidize a wide range of substrates. In an attempt to increase the process stability of CYP102A1, we exchanged the more unstable reductase domain of CYP102A1 with the more stable reductase domain of CYP102A3. Stability of the chimeric fusion protein was determined spectrophotometrically as well as by measuring the hydroxylation activity towards 12-para-nitrophenoxydodecanoic acid (12-pNCA) after incubation at elevated temperatures. In the reaction with 12-pNCA, the new chimeric protein exhibited 88 and 38% of the activity of CYP102A3 and CYP102A1, respectively, but was able to hydroxylate substrates within a wider temperature range compared with the parental enzymes. Maximum activity was obtained at 51°C, and the half-life at 50°C was with 100 min more than ten times longer than that of CYP102A1 (8 min).

Novel phacB-Encoded Cytochrome P450 Monooxygenase from Aspergillus nidulans with 3-Hydroxyphenylacetate 6-Hydroxylase and 3,4-Dihydroxyphenylacetate 6-Hydroxylase Activities

Abstract: Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (ΔphacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).

Functional activity of the mouse flavin-containing monooxygenase forms 1, 3, and 5.

Abstract: Three functional mouse flavin-containing monooxygenases (mFMOs) (i.e., mFMO1, mFMO3, and mFMO5) have been reported to be the major FMOs present in mouse liver. To examine the biochemical features of these enzymes, recombinant enzymes were expressed as maltose-binding protein fusion proteins (i.e., MBP-mFMO1, MBP-mFMO3, and MBP-mFMO5) in Escherichia coli and isolated and purified with affinity chromatography. The substrate specificity of these three mouse hepatic FMO enzymes were examined using a variety of substrates, including mercaptoimidazole, trimethylamine, S-methyl esonarimod, and an analog thereof, and a series of 10-(N,N-dimethylaminoalkyl)-2-(trifluoromethyl)phenothiazine analogs. The kinetic parameters of the three mouse FMOs for these substrates were compared in an attempt to explore substrate structure--function relationships specific for each mFMO. Utilizing a common phenothiazine substrate for all three enzymes, we compared the pH dependence for the recombinant enzymes under similar conditions. In addition, thermal stability for mFMO1, mFMO3, and mFMO5 enzymes was examined in the presence and absence of NADPH. The results revealed unique features for mFMO5, suggesting possible impact on the functional significance of this abundantly expressed FMO5 isoform in both human and mouse liver.

Dioxygen Activation at Non-Heme Diiron Centers: Oxidation of a Proximal Residue in the I100w Variant of Toluene/O-Xylene Monooxygenase Hydroxylase

Abstract: At its carboxylate-bridged diiron active site, the hydroxylase component of toluene/o-xylene monooxygenase activates dioxygen for subsequent arene hydroxylation. In an I100W variant of this enzyme, we characterized the formation and decay of two species formed by addition of dioxygen to the reduced, diiron(II) state by rapid-freeze quench (RFQ) EPR, Mossbauer, and ENDOR spectroscopy. The dependence of the formation and decay rates of this mixed-valent transient on pH and the presence of phenol, propylene, or acetylene was investigated by double-mixing stopped-flow optical spectroscopy. Modification of the α-subunit of the hydroxylase after reaction of the reduced protein with dioxygen-saturated buffer was investigated by tryptic digestion coupled mass spectrometry. From these investigations, we conclude that (i) a diiron(III,IV)−W• transient, kinetically linked to a preceding diiron(III) intermediate, arises from the one-electron oxidation of W100, (ii) the tryptophan radical is deprotonated, (iii) rapid ...

New Insights into Acetone Metabolism

Abstract: In this issue of the Journal of Bacteriology, Kotani et al. (11) elucidate the latter half of a new pathway for propane metabolism in Gordonia sp. strain TY-5, thus complementing their work on the earlier steps of the pathway from 3 years ago (10). Utilization of this gaseous three-carbon substrate, depicted in the boxed region of Fig. ​Fig.1,1, provides new insights into the microbial metabolism of acetone—a central intermediate of the overall scheme. The initial step of propane utilization involves a four-component putative di-iron monooxygenase (encoded by prnABCD) that catalyzes the NADH-dependent subterminal hydroxylation of the alkane to form 2-propanol. Three distinct NAD+-dependent secondary alcohol dehydrogenases (encoded by adh1, adh2, and adh3) convert the hydroxylated intermediate to acetone. A flavin adenine dinucleotide (FAD)-dependent monooxygenase (encoded by acmA) catalyzes an NADPH-dependent Baeyer-Villiger reaction that inserts an oxygen atom into a carbon-carbon bond of acetone to produce methyl acetate. Finally, a hydrolase (encoded by acmB) splits the ester into acetic acid and methanol. Consistent with their proposed functions, these genes are induced by propane, 2-propanol, and acetone. FIG. 1. Overview of acetone metabolism. The boxed region depicts the novel pathway of propane metabolism uncovered in Gordonia sp. strain TY-5 (10, 11). Additional reactions that generate or degrade acetone are illustrated outside of the box. The central position of acetone in the propane oxidation pathway is placed into a broader context by comparison to the other acetone-related reactions illustrated in Fig. ​Fig.1.1. Many anaerobic bacteria, such as those in the genus Clostridium (4), produce acetone during fermentation by decarboxylation of acetoacetate (reaction a). Selected plants decompose cyanogenic glucosides to generate acetone cyanohydrin, which is metabolized by a lyase (reaction b) to form hydrogen cyanide and acetone (7). Epoxypropane, formed in certain bacteria by monooxygenation of propene, can undergo isomerization (reaction c) to acetone (18). Various members of the vibrio family possess a multistep pathway of leucine catabolism that produces 3-hydroxy-3-methylglutaryl-coenzyme A (15), which decomposes to yield this ketone (reaction d). Other microbes oxidize 2-nitropropane (reaction e), atrazine (reaction f), or other compounds to produce this molecule (13, 14). In the same manner, alternative pathways for acetone decomposition have been described. One well-characterized pathway involves the ATP-dependent, manganese-containing enzyme acetone carboxylase (reaction g) that forms acetoacetate, which is subsequently converted through multiple steps to form two molecules of acetyl-coenzyme A (1, 17). The conversion of acetone to acetol by cytochrome P450 has long been known (9), and other types of monooxygenases also may be capable of catalyzing such terminal hydroxylation (scheme h). Acetol cleavage to form acetaldehyde and formaldehyde (reaction i) has been proposed (5); however, no characterization of this enzyme (presumably containing thiamine pyrophosphate to facilitate this chemistry) has been reported. Acetol dehydrogenase (reaction j) and methylglyoxal dehydrogenase (reaction k) reactions have been characterized (3) and would convert acetol to pyruvate. Finally, evidence was presented 20 years ago for an NADPH-dependent acetol monooxygenase that catalyzes Baeyer-Villiger chemistry (reaction l) in Mycobacterium sp. strain P1 (8); the resulting hydroxymethylene acetate would spontaneously decompose (reaction m) to yield acetic acid and formaldehyde. In sum, the reactions shown in Fig. ​Fig.11 highlight the position of acetone at a central metabolic crossroads. Although Baeyer-Villiger-type monooxygenases have been studied for decades, the Gordonia enzyme encoded by acmA appears to be unique in using the three-carbon compound acetone as an effective substrate. Indeed, a review of the literature reveals only slight activity with butanone as the smallest alternate substrate in one enzyme representative (2). The Baeyer-Villiger monooxygenases are flavoenzymes, and this is certainly the situation for AcmA. On the basis of its weak yellow color, this protein probably loses a portion of its FAD cofactor during purification—a situation known to occur during isolation of other family members (6, 12). Figure ​Figure22 depicts a reasonable FAD-dependent catalytic reaction for AcmA, using as a precedent the results of kinetic, mechanistic, and structural studies carried out with related enzymes. The figure is not meant to imply the order of substrate binding. For example, AcmA may exhibit a ter-ter kinetic mechanism, as described for cyclohexanone monooxygenase (16). Alternatively, the Gordonia enzyme might reduce its flavin by using NADPH, undergo a conformational change to react with oxygen, and then bind the substrate, as suggested by structural studies carried out with Thermobifida fusca phenylacetone monooxygenase (12). This structurally characterized family member is 43% identical in sequence to AcmA, raising questions concerning the structural basis of substrate specificity. Unfortunately, no structure is available for a substrate-bound form of any Baeyer-Villiger monooxygenase. Figure ​Figure33 depicts a surface representation of the T. fusca protein with its flavin illustrated in yellow. The residues lining its substrate-binding pocket are compared to the Gordonia enzyme and shown in green if conserved (Asp66, Leu153, Arg217, Thr218, Tyr331, Arg337, Gly388, Phe389, Ala391, and Ser500) or in other coloring when substituted (C65F, blue; S196A, teal; H220Q, magenta; K336H, cyan; I339P, red). Further comparisons of the sequences indicate a single amino acid deletion in AcmA, corresponding to Thr256 of the T. fusca enzyme, and three single amino acid insertions in the Gordonia protein (between Ile288 and Leu289, Glu375 and Arg376, and Ser441 and Ala442 of the phenylacetone monooxygenase). The first three of these changes are distant from the active site and unlikely to be important to substrate specificity or catalysis, whereas the latter insertion is predicted to occur near the FAD. Additional kinetic studies are needed to determine the catalytic efficiencies of the various substrates of AcmA, and mutagenic or structural investigations are required to discern the structural basis of the substrate specificity of these enzymes. FIG. 2. Mechanism of acetone monooxygenase AcmA, a Baeyer-Villiger-type monooxygenase. The order of substrate binding and product release is unknown; however, the reduced nicotinamide must be used to first reduce the flavin, the reduced flavin then reacts with ... FIG. 3. Surface representation of phenylacetone monooxygenase and comparison of residues lining its substrate-binding pocket with those of AcmA. The structure of T. fusca phenylacetone monooxygenase (Protein Data Bank access code 1w4x) is shown in a surface representation ...

Enantiocomplementary preparation of (S)- and (R)-mandelic acid derivatives via α-hydroxylation of 2-arylacetic acid derivatives and reduction of α-ketoester using microbial whole cells

Abstract: Forty one microorganisms belonging to different taxonomical groups were used to carry out the enantioselective reduction of methyl benzoylformate to afford the corresponding (R)-methyl mandelate, with moderate to high ee. In contrast, the monooxygenase enzyme in Helminthosporium sp. CIOC3.3316 catalyzed the hydroxylation of methyl 2-phenylacetate to (S)-methyl mandelate. This combination of oxidation and reduction biotransformations thus provides a method for preparing the enantiomers of chiral α-hydroxy acid derivatives.

Design of a secondary alcohol degradation pathway from Pseudomonas fluorescens DSM 50106 in an engineered Escherichia coli

Abstract: The genes encoding an alcohol dehydrogenase, Baeyer–Villiger monooxygenase and an esterase from P. fluorescens DSM 50106, which seemed to be metabolically connected based on the sequence of the corresponding open reading frames, were cloned into one vector (pABE) and functionally expressed in Escherichia coli. Overall expression levels were quite low, however, using whole cells of E. coli JM109 pABE expressing the three recombinant enzymes, conversion of secondary alcohols (Cn) to the corresponding primary alcohols (Cn−2) and acetic acid via ketone and ester was possible. In this way, 2-decanol was almost completely converted within 20 h at 30°C. Thus, it could be shown that the three enzymes are metabolically connected and that they are most probably involved in alkane degradation via sub-terminal oxidation of the acyclic aliphatic hydrocarbons.

Epoxyeicosatrienoic acids induce growth inhibition and calpain/caspase-12 dependent apoptosis in PDGF cultured 3T6 fibroblast.

Abstract: Previous studies have demonstrated that arachidonic acid (AA) metabolites released by the cyclooxygenase pathway is involved in serum-induced 3T6 fibroblast cycle progression and proliferation. However, these results also suggest that other AA cascade pathways might be involved. Recently, we also described the role of hydroxyeicosatetraenoic acids, which are produced by cytochrome P450 monooxygenases (CYP), in 3T6 fibroblast growth. AA can be also metabolized by the epoxygenase activity of CYP-producing epoxyeicosatrienoic acids (EETs). Finally, the cytosolic epoxide hydrolases catalyze the hydration of the EETs, transforming them into dihydroxyeicosatetraenoic acids (DHETEs). In this work, we have studied the role of the EETs/DHETEs on 3T6 fibroblasts growth. Our results show that PDGF stimulates 3T6 fibroblast proliferation and [3H]thymidine incorporation, while the addition of 5,6-EET, 8,9-EET, 11,12-EET or 14,15-EET (0.1-1 microM) inhibit these processes. Furthermore, 5,6-DHETE and 11,12-DHETE (0.1-1 microM) also inhibit cell proliferation and DNA synthesis. Interestingly, this growth inhibition was correlated with an induction of apoptosis. Thus, we observed that in the presence of PDGF, EETs or DHETEs (0.1-1 microM) induce phosphatidylserine externalization (as measured by annexin V-binding) and DNA fragmentation (as quantified using a TUNEL assay). Our results show that calpain, as well as caspase-12 and caspase-3, are involved in these events. Therefore, EETs and DHETEs have anti-proliferative and pro-apoptotic effects on PDGF-stimulated 3T6 fibroblasts.

Growth of Aquincola tertiaricarbonis L108 on tert‐Butyl Alcohol Leads to the Induction of a Phthalate Dioxygenase‐related Protein and its Associated Oxidoreductase Subunit

Abstract: The microbial degradation of tert-butyl alcohol (TBA), an important environmental pollutant and an intermediate in the degradation of methyl tert-butyl ether (MTBE), was proposed to involve a monooxygenase for the initial oxidation of TBA, but up to now a specific enzyme with that activity has not been described except the well-known AlkB for the Gram-positive strain Mycobacterium austro-africanum IFP2012 (Lopez Ferreira et al., Appl. Microbiol. Biotechnol. 2007, 75, 909-919). In the course of our studies of the MTBE pathway, the proteome patterns in one- and two-dimensional gels of Aquincola tertiaricarbonis L108 which was grown on lactate, on hydroxyisobutyrate (2-HIBA) and TBA, were compared. A protein of about 55 kDa was detected after growth on TBA and 2-HIBA, which, after mass spectrometric analysis of the tryptic digested peptides, was assigned with a high score to phthalate dioxygenase. Sequence analysis of PCR products obtained with primers derived from the amino acid sequences in the above peptides supported the assignment to the hydroxylase subunit of phthalate dioxygenase-like proteins by covering 96.7% of a corresponding gene from Methylibium petroleiphilum PM1. The conserved amino acid motifs -R-x 12 -CxHRxxxLxxG-X 8 -CxYHR-X 6 -G- for the Rieske [2Fe-2S] binding domain and (-D/E)xxxDxxHxxxxH- for the mononuclear iron binding domain were found. A second protein of about 38 kDa was detected after growth on TBA with a lower score and attributed to a putative iron-sulfur oxidoreductase subunit. Primers derived from the peptides resulted in an amplicon, which covered 75.7 % of a corresponding gene from M. petroleiphilum PM1. Conserved motifs -RxYSL-x 20-22 -RGGS- for FMN binding and -GGIGxTPxxxM- for NAD binding were detected, which suggests that this protein is the small subunit of a two-component phthalate dioxygenase-like enzyme typically containing FMN. Dioxygenase-related enzymes are known to catalyze also monooxygenase reactions (see e.g. Zhou et al. J. Bacterial. 2002, 184, 1547-1555), which makes it likely that the two proteins induced in the presence of TBA are involved in TBA oxidation.