Biodegradation of fuel oxygenates and their effect on the expression of a newly identified cytochrome P450 gene in Achromobacter xylosoxidans MCM2/2/1

Achromobacter xylosoxidans MCM2/2/1 was enriched and isolated from gasoline-contaminated soil and was found to degrade ethyl tert-butyl ether (ETBE) and methyl tert-butyl ether (MTBE) by 41.48% and 34.15%, respectively, in 6 days. Furthermore, the effect of MTBE and TBA on the expression of cytochrome P450 (CYP) of A. xylosoxidans MCM2/2/1 was examined. The presence of the CYP gene in this organism was first confirmed by amplification of a putative 350 bp CYP gene fragment followed by identification of the entire gene by genome walking and DNA-sequencing. The identified CYP gene of A. xylosoxidans MCM2/2/1 shares a high similarity of about 88% with the thcB gene of A. xylosoxidans A8. Gene expression studies have shown that the CYP gene is expressed in A. xylosoxidans MCM2/2/1; however, the expression of this gene was altered at different concentrations of MTBE.     

The cytochrome P450 genes of channel catfish: Their involvement in disease defense responses as revealed by meta-analysis of RNA-Seq data sets

Background

Cytochrome P450s (CYPs) encode one of the most diverse enzyme superfamily in nature. They catalyze oxidative reactions of endogenous molecules and exogenous chemicals.

Methods

We identified CYPs genes through in silico analysis using EST, RNA-Seq and genome databases of channel catfish. Phylogenetic analyses and conserved syntenic analyses were conducted to determine their identities and orthologies. Meta-analysis of RNA-Seq databases was conducted to analyze expression profile of CYP genes following bacterial infection.

Results

A full set of 61 CYP genes was identified and characterized in channel catfish. Phylogenetic tree and conserved synteny provided strong evidence of their identities and orthorlogy. Lineage-specific gene duplication was evident in a number of clans in channel catfish. CYP46A1 is missing in the catfish genome as observed with syntenic analysis and RT-PCR analysis. Thirty CYPs were found up- or down-regulated in liver, while seven and eight CYPs were observed regulated in intestine and gill following bacterial infection.

Conclusion

We systematically identified and characterized a full set of 61 CYP genes in channel catfish and studied their expression profiles after bacterial infection. While bacterial challenge altered the expression of large numbers of CYP genes, the mechanisms and significance of these changes are not known.

General significance

This work provides an example to systematically study CYP genes in non-model species. Moreover, it provides a basis for further toxicological and physiological studies in channel catfish.     

The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution.     

Differential metabolism of diastereoisomeric diterpenes by Preussia minima,found as endophytic fungus in Cupressus lusitanica

The plant diastereoisomeric diterpenes ent-pimara-8(14)-15-dien-19-oic acid, obtained from Viguiera arenaria, and isopimara-8(14)-15-dien-18-oic acid, isolated from Cupressus lusitanica, were distinctly functionalized by the enzymes produced in whole cell cultures of the fungus Preussia minima, isolated from surface sterilized stems of C. lusitanica. The ent-pimaradienoic acid was transformed into the known 7β-hydroxy-ent-pimara-8(14)-15-dien-19-oic acid, and into the novel diterpenes 7-oxo-8 β-hydroxy-ent-pimara-8(14)-15-dien-19-oic and 7-oxo-9β-hydroxy-ent-pimara-8(14)-15-dien-19-oic acids. Isopimara-8(14)-15-dien-18-oic acid was converted into novel diterpenes 11α-hydroxyisopimara-8(14)-15-dien-18-oic acid, 7β,11α-dihydroxyisopimara-8(14)-15-dien-18-oic acid, and 1β,11α-dihydroxyisopimara-8(14)-15-dien-18-oic acid, along with the known 7β-hydroxyisopimara-8(14)-15-dien-18-oic acid. All compounds were isolated and fully characterized by 1D and 2D NMR, especially ¹³C NMR. The diterpene bioproduct 7-oxo-9β-hydroxy-ent-pimara-8(14)-15-dien-19-oic acid is an isomer of sphaeropsidin C, a phytotoxin that affects cypress trees produced by Shaeropsis sapinea, one of the main phytopathogen of Cupressus. The differential metabolism of the diterpene isomers used as substrates for biotransformation was interpreted with the help of computational molecular docking calculations, considering as target enzymes those of cytochrome P450 group.     

Cellular retinoid-binding proteins transfer retinoids to human cytochrome P450 27C1 for desaturation

Cytochrome P450 27C1 (P450 27C1) is a retinoid desaturase expressed in the skin that catalyzes the formation of 3,4-dehydroretinoids from all-trans retinoids. Within the skin, retinoids are important regulators of proliferation and differentiation. In vivo, retinoids are bound to cellular retinol-binding proteins (CRBPs) and cellular retinoic acid–binding proteins (CRABPs). Interaction with these binding proteins is a defining characteristic of physiologically relevant enzymes in retinoid metabolism. Previous studies that characterized the catalytic activity of human P450 27C1 utilized a reconstituted in vitro system with free retinoids. However, it was unknown whether P450 27C1 could directly interact with holo-retinoid-binding proteins to receive all-trans retinoid substrates. To assess this, steady-state kinetic assays were conducted with free all-trans retinoids and holo-CRBP-1, holo-CRABP-1, and holo-CRABP-2. For holo-CRBP-1 and holo-CRABP-2, the k_cat/K_m values either decreased 5-fold or were equal to the respective free retinoid values. The k_cat/K_m value for holo-CRABP-1, however, decreased ∼65-fold in comparison with reactions with free all-trans retinoic acid. These results suggest that P450 27C1 directly accepts all-trans retinol and retinaldehyde from CRBP-1 and all-trans retinoic acid from CRABP-2, but not from CRABP-1. A difference in substrate channeling between CRABP-1 and CRABP-2 was also supported by isotope dilution experiments. Analysis of retinoid transfer from holo-CRABPs to P450 27C1 suggests that the decrease in k_cat observed in steady-state kinetic assays is due to retinoid transfer becoming rate-limiting in the P450 27C1 catalytic cycle. Overall, these results illustrate that, like the CYP26 enzymes involved in retinoic acid metabolism, P450 27C1 interacts with cellular retinoid-binding proteins.     

Effects of Culture Conditions on Production of trans-Cinnamic Acid from Alkylbenzenes by Soil Microorganisms

The production of trans-cinnamic acid from various alkylbenzenes by soil microorganisms was studied intensively by use of a co-oxidation technique. The microorganisms were grown on n-paraffins, and they did not use aromatic compounds as a carbon source when the preferred substrate was present in the medium. The effects of cell population, co-oxidation time, and type and mode of addition of the alkylbenzenes on the yield of trans-cinnamic acid were investigated. Yields (5 g/liter) of a product consisting of trans-cinnamic acid (88 to 100%) and 5-phenylvaleric acid (0 to 12%) were obtained when the proper conditions were chosen. Of a variety of microorganisms studied, a soil isolate closely related to Cellulomonas galba was found to be best for the production of trans-cinnamic acid.     

Altered Expression of Small Heterodimer Partner Governs Cytochrome P450 (CYP) 2D6 Induction during Pregnancy in CYP2D6-humanized Mice

Substrates of a major drug-metabolizing enzyme CYP2D6 display increased elimination during pregnancy, but the underlying mechanisms are unknown in part due to a lack of experimental models. Here, we introduce CYP2D6-humanized (Tg-CYP2D6) mice as an animal model where hepatic CYP2D6 expression is increased during pregnancy. In the mouse livers, expression of a known positive regulator of CYP2D6, hepatocyte nuclear factor 4α (HNF4α), did not change during pregnancy. However, HNF4α recruitment to CYP2D6 promoter increased at term pregnancy, accompanied by repressed expression of small heterodimer partner (SHP). In HepG2 cells, SHP repressed HNF4α transactivation of CYP2D6 promoter. In transgenic (Tg)-CYP2D6 mice, SHP knockdown led to a significant increase in CYP2D6 expression. Retinoic acid, an endogenous compound that induces SHP, exhibited decreased hepatic levels during pregnancy in Tg-CYP2D6 mice. Administration of all-trans-retinoic acid led to a significant decrease in the expression and activity of hepatic CYP2D6 in Tg-CYP2D6 mice. This study provides key insights into mechanisms underlying altered CYP2D6-mediated drug metabolism during pregnancy, laying a foundation for improved drug therapy in pregnant women.     

Comparative functional characterization of a novel benzoate hydroxylase cytochrome P450 of Fusarium oxysporum

FoCYP53A19, a novel cytochrome P450 capable of performing benzoate hydroxylation, was identified and characterized from the ascomycete Fusarium oxysporum f.sp. lycopersici. Comparative functional analysis of FoCYP53A19 with the heterologous and homologous cytochrome P450 reductases (CPR) such as Saccharomyces cerevisiae (ScCPR), Candida albicans (CaCPR) and F. oxysporum (FoCPR) revealed novel catalytic properties. The catalytic efficiency and substrate specificity of FoCYP53A19 were significantly influenced and altered by the source of the reductase employed. The yeast reconstitution system of FoCYP53A19 with ScCPR performed the hydroxylation of benzoic acid (BA) and demethylation of 3-methoxybenzoic acid (3-MBA); but when reconstituted with CaCPR, FoCYP53A19 performed only the essential hydroxylation of fungal benzoate catabolism. Remarkably, FoCYP53A19 with its homologous reductase FoCPR, not only demonstrated the improved conversion rates of BA and 3-MBA, but also exhibited activity toward the hydroxylation of 3-hydroxybenzoic acid. The electron transfer compatibility and the coupling efficiency between the homologous FoCYP-FoCPR system are significant and it favored enhanced monooxygenase activity with broader substrate specificity.     

A spectrophotometric assay for trans-cinnamic acid 4-hydroxylase activity.

trans-Cinnamic acid 4-hydroxylase (trans-cinnamic acid, NADPH: oxygen oxidoreductase [4-hydroxylating]) can be rapidly and precisely assayed by the spectrophotometric measurement of the production of 4-hydroxy-trans-cinnamic acid at 340 nm after acidification of the reaction mixture and subsequent readjustment to pH 11. For the assay of crude extracts and other preparations with high intrinsic absorption at 340 nm, the assay can be modified by extraction of the 4-hydroxy-trans-cinnamic acid from the acidified assay mixture through diethyl ether into alkali before spectrophotometric estimation. trans-Cinnamic acid 2-hydroxylase can be routinely detected and assayed in the same extract.     

Origins of P450 diversity

The P450 enzymes maintain a conserved P450 fold despite a considerable variation in sequence. The P450 family even includes proteins that lack the single conserved cysteine and are therefore no longer haem-thiolate proteins. The mechanisms of successive gene duplications leading to large families in plants and animals are well established. Comparisons of P450 CYP gene clusters in related species illustrate the rapid changes in CYPome sizes. Examples of CYP copy number variation with effects on fitness are emerging, and these provide an opportunity to study the proximal causes of duplication or pseudogenization. Birth and death models can explain the proliferation of CYP genes that is amply illustrated by the sequence of every new genome. Thus, the distribution of P450 diversity within the CYPome of plants and animals, a few families with many genes (P450 blooms) and many families with few genes, follows similar power laws in both groups. A closer look at some families with few genes shows that these, often single member families, are not stable during evolution. The enzymatic prowess of P450 may predispose them to switch back and forth between metabolism of critical structural or signal molecules and metabolism dedicated to environmental response.     

Metabolism of lunularic acid in liverworts

The presence, in Conocephalum conicum, of the enzymes phenylalanine ammonia lyase, trans-cinnamic acid-4-hydroxylase and lunularic acid decarbo     

Characterization of hsr201 and hsr515, two tobacco genes preferentially expressed during the hypersensitive reaction provoked by phytopathogenic bacteria

During an incompatible interaction between tobacco and the bacterial phytopathogen Pseudomonas solanacearum, 2 classes of genes, the so-called hsr (hypersensitivity-related) genes, activated preferentially during the hypersensitive reaction, and the str (sensitivity-related) genes, expressed strongly during compatible and incompatible interactions, have been identified. In this report, two hsr cDNA clones, hsr515 and hsr201, as well as their expression patterns are presented. Hsr515 was found to encode a P450 monooxygenase and is most similar to the ripening-related avocado gene CYP71A1 (40.6% amino acid identity). Hsr201 presents 58.6% amino acid identity with pTom36, a tomato gene expressed during fruit maturation. The putative functions of the hsr gene products appear to be quite diverse and their characteristics of activation were found to be very conserved: accumulation of the corresponding mRNAs primarily in leaf areas in contact with the avirulent P. solanacearum strain or with a Pseudomonas fluorescens strain containing the hrpZ gene encoding a necrotizing polypeptide, harpin and absence of expression during normal plant development. Our results also suggest that, in a tobacco line expressing NahG, a lower level of salicylic acid, a compound associated with systemic acquired resistance, and also possibly involved in the development of necrotic lesions characteristic of the HR, does not affect the hsr gene expression.     

Cloning and expression analysis of the cytochrome P450c17s enzymes during the reproductive cycle in ovoviviparous Korean rockfish (Sebastes schlegeli)

Cytochrome P450c17 (CYP17, 17α-hydroxylase/17, 20-lyase) plays a critical role in the production of androgens and estrogens in vertebrates. We isolated the full length cDNAs of P450c17-I and P450c17-II from Sebastes schlegeli. The cDNA sequences of P450c17-I and P450c17-II encoded 515 and 533 amino acid residues respectively. The putative P450c17-I and P450c17-II enzymes of Korean rockfish share high sequence identity with that of Japanese flounder (92% and 81%) respectively. Our current study describes that P450c17s of Korean rockfish are mainly expressed in gonads, head kidney and kidney by RT-PCR. Quantitative real-time PCR showed that the expression patterns of Korean rockfish P450c17s were developmental stage-dependency. In addition, the testosterone (T) and gonadosomatic index (GSI) levels further support the important role of P450c17-I during shift in steroidogenesis. Taken together, this study provides information about the Korean rockfish P450c17s characterization and mRNA expression as such helps in further understanding of its function in gonadal development.     

Regulation of phenylpropanoid metabolism by exogenous precursors in axenic cultures of Sphagnum fallax

Sphagnum plantlets, cultivated in continuous-feed bioreactors, are characterised by high levels of free endogenous phenolics and a pronounced excretion of some phenolics into the effluent culture medium. The transfer of Sphagnum fallax, precultivated in continuous-feed bioreactors, to batch cultures resulted in an increased flux through phenylpropanoid metabolism and an accumulation of p-coumaric acid to 0.1 μM and of trans-sphagnum acid up to 0.5 μM in the external medium [³H]-labelled L-phenylalanine (7.7 GBq mol^?1) was rapidly taken up, resulting in an enhanced synthesis and excretion of p-coumaric and trans-sphagnum acid. Specific activities were 6.9 and 5.4 GBq mol^?1, respectively, for these cinnamic acids excreted into the external medium. Endogenous pools of trans-cinnamic and p-coumaric acid did not increase and no labelling could be detected in these compounds. Cell wall-bound activity amounted to ca 14% of the applied activity after 48 h of incubation, 59% of which was recovered in dioxane/2 M HCl extracts of the cell wall. Exogenously applied trans-cinnamic acid (0.1 mM) was taken up to 46% and resulted in a transient endogenous accumulation of trans-cinnamic acid, the level of free endogenous p-coumaric and trans-sphagnum acid was found to have decreased. The concentrations of p-coumaric and trans-sphagnum acid in the culture medium rose to 17 and 2.4 μM, respectively, after 48 h of incubation in 0.1 mMtrans-cinnamic acid. Exogenously applied p-coumaric acid (0.1 mM) was taken up to 79% from the incubation solution but not stored endogenously, as metabolic products trans-sphagnum acid and an unknown p-coumaric acid-conjugate accumulated in the external medium and endogenously. These results give evidence for the biosynthetical route from phenylalanine to sphagnum acid and a channelling of pathway intermediates by the enzymes L-phenylalanine ammonia-lyase (EC 4.3.1.5) and cinnamic acid 4-hydroxylase (EC 1.14.13.11).     

Phenylacetic acid meta hydroxylase from Rhizoctonia solani

Mono-oxygenase-like activity occurs in Rhizoctonia solani during the metabolism of phenyl-acetic acid. The partially purified enzyme catalysed only the hydroxylation of phenylacetic acid at its meta position and required NADH and tetrahydrofolate as co-factors. Benzoic, phenylpropionic, trans-cinnamic, and phenoxyacetic acids were not suitable substrates for the enzyme.     

Cytochrome P450-dependent metabolism of eicosapentaenoic acid in the nematode Caenorhabditis elegans

The genome of Caenorhabditis elegans contains 75 full length cytochrome P450 (CYP) genes whose individual functions are largely unknown yet. We tested the hypothesis that some of them may be involved in the metabolism of eicosapentaenoic acid (EPA), the predominant polyunsaturated fatty acid of this nematode. Microsomes isolated from adult worms contained spectrally active CYP proteins and showed NADPH-CYP reductase (CPR) activities. They metabolized EPA and with lower activity also arachidonic acid (AA) to specific sets of regioisomeric epoxy- and ω-/(ω-1)-hydroxy-derivatives. 17(R),18(S)-epoxyeicosatetraenoic acid was produced as the main EPA metabolite with an enantiomeric purity of 72%. The epoxygenase and hydroxylase reactions were NADPH-dependent, required the functional expression of the CPR-encoding emb-8 gene, and were inhibited by 17-ODYA and PPOH, two compounds known to inactivate mammalian AA-metabolizing CYP isoforms. Multiple followed by single RNAi gene silencing experiments identified CYP-29A3 and CYP-33E2 as the major isoforms contributing to EPA metabolism in C. elegans. Liquid chromatography/mass spectrometry revealed that regioisomeric epoxy- and hydroxy-derivatives of EPA and AA are endogenous constituents of C. elegans. The endogenous EPA metabolite levels were increased by treating the worms with fenofibrate, which also induced the microsomal epoxygenase and hydroxylase activities. These results demonstrate for the first time that C. elegans shares with mammals the capacity to produce CYP-dependent eicosanoids and may thus facilitate future studies on the mechanisms of action of this important class of signaling molecules.     

Characterization of two NADPH: Cytochrome P450 reductases from cotton (Gossypium hirsutum)

Cytochrome P450 monooxygenases (P450s) are commonly involved in biosynthesis of endogenous compounds and catabolism of xenobiotics, and their activities rely on a partner enzyme, cytochrome P450 reductase (CPR, E.C.1.6.2.4). Two CPR cDNAs, GhCPR1 and GhCPR2, were isolated from cotton (Gossypium hirsutum). They are 71% identical to each other at the amino acid sequence level and belong to the Class I and II of dicotyledonous CPRs, respectively. The recombinant enzymes reduced cytochrome c, ferricyanide and dichlorophenolindophenol (DCPIP) in an NADPH-dependent manner, and supported the activity of CYP73A25, a cinnamate 4-hydroxylase of cotton. Both GhCPR genes were widely expressed in cotton tissues, with a reduced expression level of GhCPR2 in the glandless cotton cultivar. Expression of GhCPR2, but not GhCPR1, was inducible by mechanical wounding and elicitation, indicating that the GhCPR2 is more related to defense reactions, including biosynthesis of secondary metabolites.