Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbons n-alkanes. A homology-based 3D-model revealed presence of an extraordinarily large active site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2 and CYP1B1) and bacterial aliphatic hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of varying ring size, including 4 rings (pyrene and fluoranthene), 5 rings [lsqb]benzo(a)pyrene[rsqb], and 6 rings [lsqb]benzo(ghi)perylene[rsqb]; the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane ω-hydroxylase that oxidized varying chain-length n-alkanes (C9-C12 and C15-C19) as well as alkyl side-chains (C3-C9) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long chain-length APs and alkanes. To our knowledge, this is the first of its kind P450 across biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills).
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"Direct experimental evidence is necessary to validate this idea. Cytochrome P450 monooxygenases are alternative biocatalysts for the oxidation of aliphatic and aromatic hydrocarbons in many hydrocarbon-degrading microorganisms (van Beilen et al. 2006; Syed et al. 2013). Altogether, 16 cyp genes were found in R. erythropolis PR4. "
[Show abstract][Hide abstract] ABSTRACT: Rhodococcus erythropolis PR4 is able to degrade diesel oil, normal-, iso- and cycloparaffins and aromatic compounds. The complete DNA content of the strain was previously sequenced and numerous oxygenase genes were identified. In order to identify the key elements participating in biodegradation of various hydrocarbons, we performed a comparative whole transcriptome analysis of cells grown on hexadecane, diesel oil and acetate. The transcriptomic data for the most prominent genes were validated by RT-qPCR. The expression of two genes coding for alkane-1-monooxygenase enzymes was highly upregulated in the presence of hydrocarbon substrates. The transcription of eight phylogenetically diverse cytochrome P450 (cyp) genes was upregulated in the presence of diesel oil. The transcript levels of various oxygenase genes were determined in cells grown in an artificial mixture, containing hexadecane, cycloparaffin and aromatic compounds and six cyp genes were induced by this hydrocarbon mixture. Five of them were not upregulated by linear and branched hydrocarbons. The expression of fatty acid synthase I genes was downregulated by hydrocarbon substrates, indicating the utilization of external alkanes for fatty acid synthesis. Moreover, the transcription of genes involved in siderophore synthesis, iron transport and exopolysaccharide biosynthesis was also upregulated, indicating their important role in hydrocarbon metabolism. Based on the results, complex metabolic response profiles were established for cells grown on various hydrocarbons. Our results represent a functional annotation of a rhodococcal genome, provide deeper insight into molecular events in diesel/hydrocarbon utilization and suggest novel target genes for environmental monitoring projects.
"Recent studies on fungal organisms revealed the presence of a large number of P450 contingents in their genomes, with some exceptions , , . Subsequent functional studies suggested that fungal P450s are catalytically diverse , ,  and perform extraordinary oxidation activity compared to P450s across the biological kingdoms . The latest study conducted in our laboratory revealed the presence of a large number of thermostable P450s with biotechnological potential in fungi . "
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins distributed across the biological kingdoms. P450s are catalytically versatile and play key roles in organisms primary and secondary metabolism. Identification of P450s across the biological kingdoms depends largely on the identification of two P450 signature motifs, EXXR and CXG, in the protein sequence. Once a putative protein has been identified as P450, it will be assigned to a family and subfamily based on the criteria that P450s within a family share more than 40% homology and members of subfamilies share more than 55% homology. However, to date, no evidence has been presented that can distinguish members of a P450 family. Here, for the first time we report the identification of EXXR- and CXG-motifs-based amino acid patterns that are characteristic of the P450 family. Analysis of P450 signature motifs in the under-explored fungal P450s from four different phyla, ascomycota, basidiomycota, zygomycota and chytridiomycota, indicated that the EXXR motif is highly variable and the CXG motif is somewhat variable. The amino acids threonine and leucine are preferred as second and third amino acids in the EXXR motif and proline and glycine are preferred as second and third amino acids in the CXG motif in fungal P450s. Analysis of 67 P450 families from biological kingdoms such as plants, animals, bacteria and fungi showed conservation of a set of amino acid patterns characteristic of a particular P450 family in EXXR and CXG motifs. This suggests that during the divergence of P450 families from a common ancestor these amino acids patterns evolve and are retained in each P450 family as a signature of that family. The role of amino acid patterns characteristic of a P450 family in the structural and/or functional aspects of members of the P450 family is a topic for future research.
"Recent genome sequencing of fungal species revealed the presence of a large number of P450s in their genomes, with some exceptions (Moktali et al. 2012; Nelson 2011). Functional analysis of fungal P450s showed the presence of P450s displaying extraordinary catalytic activities (Syed et al. 2013b) and recently a fungal P450 has been engineered to oxidize carcinogenic and/or mutagenic compounds (Syed et al. 2013c). However, to date, P450s with a thermostable nature have not been reported from this kingdom. "
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 monooxygenases (P450s) are ubiquitous heme-thiolate proteins that have potential biotechnological application. Thermostable-P450s that can withstand hostile industrial conditions, such as high temperatures, extremes of pH and organic solvents, are needed for biotechnological usage. Here, for the first time, we report a large number of thermostable-P450s from two thermophilic biomass-degrading fungi, Myceliophthora thermophila and Thielavia terrestris. Genome-wide P450 analysis revealed the presence of 79 and 70 P450s (P450ome) in T. terrestris and M. thermophila. Authentic P450s containing both the P450 signature domains (EXXR and CXG) were classified as follows: T. terrestris (50 families and 56 subfamilies) and M. thermophila (49 families and 53 subfamilies). Bioinformatics analysis of P450omes suggested the presence of a large number of thermostable-P450s. Based on aliphatic index cut-off (>90), 14 and 11 P450s were determined to be thermostable in T. terrestris and M. thermophila. Among the thermostable P450s, six P450s from T. terrestris and three from M. thermophila had a melting temperature (Tm) of >65 °C, suggesting their hyperthermal tolerance. Analysis of the instability index of two ascomycete P450omes revealed the presence of 12 and 19 in vitro stable P450s in T. terrestris and M. thermophila. Overall, six P450s from T. terrestris and four from M. thermophila showed both thermal tolerance and in vitro stability. Thermophilic ascomycetes P450s are of potential interest from a structural, mechanistic and biotechnological point of view, as five P450s showed higher thermal tolerance and five showed higher in vitro stability compared to the well-characterized thermostable-P450s CYP175A1 (bacteria) and CYP119 (archaea).