Search Results (39)

Caleosin/peroxygenases (CLO/PXGs) play critical functional roles during plant development, oxylipin metabolism, and the response to abiotic/biotic stressors and environmental toxins. In Oryza sativa, peroxygenase-9 (OsPXG9) catabolizes intermediates in oxylipin biosynthesis produced by lipoxygenase-9 (9-LOX) and scavenges HOOH and CuOOH by transferring oxygen to hydroxy fatty acids (HFAs) but not to the free fatty acids. The resulting epoxide derivatives of HFAs are then enzymatically or non-enzymatically hydrolyzed into the corresponding trihydroxy derivatives. Results presented here demonstrate OsPXG9′s specificity for catabolizing products of the 9-LOX (and not for the 13-LOX) pathway of oxylipin biosynthesis. Overexpression of OsPXG9 reduces ROS (reactive oxygen species) abundance and reduces drought- and salt-stress-induced apoptotic cell death. The high expression level of OsPXG9 also stimulates drought- and salt-induced but not basal expression of antioxidant enzymes/pathways in plants, thereby increasing cellular resistance to drought. These results suggest that OsPXG9 decreases ROS abundance and is essential to increase resilience in rice plants exposed to exogenous or endogenous abiotic stress. Full article

The cytochrome P450 family consists of ubiquitous monooxygenases with the potential to perform a wide variety of catalytic applications. Among the members of this family, CYP116B5hd shows a very prominent resistance to peracid damage, a property that makes it a promising tool for fine chemical synthesis using the peroxide shunt. In this meticulous study, we use hyperfine spectroscopy with a multifrequency approach (X- and Q-band) to characterize in detail the electronic structure of the heme iron of CYP116B5hd in the resting state, which provides structural details about its active site. The hyperfine dipole–dipole interaction between the electron and proton nuclear spins allows for the locating of two different protons from the coordinated water and a beta proton from the cysteine axial ligand of heme iron with respect to the magnetic axes centered on the iron. Additionally, since new anti-cancer therapies target the inhibition of P450s, here we use the CYP116B5hd system—imidazole as a model for studying cytochrome P450 inhibition by an azo compound. The effects of the inhibition of protein by imidazole in the active-site geometry and electron spin distribution are presented. The binding of imidazole to CYP116B5hd results in an imidazole–nitrogen axial coordination and a low-spin heme Fe^III. HYSCORE experiments were used to detect the hyperfine interactions. The combined interpretation of the gyromagnetic tensor and the hyperfine and quadrupole tensors of magnetic nuclei coupled to the iron electron spin allowed us to obtain a precise picture of the active-site geometry, including the orientation of the semi-occupied orbitals and magnetic axes, which coincide with the porphyrin N-Fe-N axes. The electronic structure of the iron does not seem to be affected by imidazole binding. Two different possible coordination geometries of the axial imidazole were observed. The angles between g_x (coinciding with one of the N-Fe-N axes) and the projection of the imidazole plane on the heme were determined to be −60° and −25° for each of the two possibilities via measurement of the hyperfine structure of the axially coordinated ¹⁴N. Full article

Eighteen short- to medium-chain monounsaturated fatty acids were screened for hydroxylation and epoxidation using eleven different peroxygenase preparations. Most of these unspecific peroxygenases (UPOs) are secreted by fungal species of the dark-spored basidiomycetous families Psathyrellaceae and Strophariaceae, two belonged to the white-spored genus Marasmius (Marasmiaceae), and one belonged to the ascomycetous family Chaetomiaceae. The fatty acids (FAs) studied were categorized into three groups based on the position of the double bond: (i) terminal unsaturated FAs (between ω and ω-1), (ii) α-β-unsaturated FAs (between C2 and C3), and (iii) β-γ-unsaturated FAs (between C3 and C4). Their chain lengths ranged from three to nine carbon atoms. FAs with a terminal double bond were significantly oxidized by only two UPOs, namely CglUPO and CraUPO (peroxygenases from Chaetomium globosum and Coprinellus radians, respectively), producing different products. FAs with internal double bonds were converted by all tested UPOs. While epoxides were observed as products in the case of α-β-unsaturated fatty acids, only CglUPO formed β-γ-epoxides from the corresponding FAs. The product pattern of the other UPOs for β-γ-unsaturated FAs was quite similar. On the other hand, the product pattern for oxidized α-β-unsaturated FAs was more variable and, in some cases, specific to a particular UPO. For example, in the reaction with trans-2-nonenoic acid, the enzymes clustered into six groups based on the formed products. Full article

Biocatalyzed oxidations are an important target in sustainable synthesis since chemical oxidations often require harsh conditions and metal-based catalysts. A raw peroxygenase-containing enzymatic preparation from oat flour was tested as a biocatalyst for the enantioselective oxidation of sulfides to sulfoxides and the variations of some reaction parameters were evaluated. Under optimal conditions, thioanisole was fully converted into the corresponding (R)-sulfoxide with high optical purity (80% ee) and the same stereopreference was maintained in the oxidation of some other sulfides. Changes in the substituent on the sulfur atom affected the selectivity of the enzyme and the best results were obtained with phenyl methoxymethyl sulfide, which gave the corresponding sulfoxide in 92% ee as exclusive product. The over-oxidation of sulfides to sulfones was instead detected in all the other cases and preferential oxidation of the (S)-enantiomer of the sulfoxide intermediate was observed, albeit with low selectivity. Carrying out the oxidation of thioanisole up to the 29% formation of sulfone led to enhancement of the sulfoxide optical purity (89% ee). The activity in sulfoxidation reactions, in addition to that reported in the epoxidation of different substrates, makes this plant peroxygenase a promising and useful tool in organic synthesis. Full article

Multi-contamination by organic pollutants and toxic metals is common in anthropogenic and industrial environments. In this study, the five fungal strains Chaetomium jodhpurense (MH667651.1), Chaetomium maderasense (MH665977.1), Paraconiothyrium variabile (MH667653.1), Emmia lacerata, and Phoma betae (MH667655.1), previously isolated in Tunisia, were investigated for the simultaneous removal and detoxification of phenanthrene (PHE) and benzo[a]anthracene (BAA), as well as heavy metals (HMs) (Cu, Zn, Pb and Ag) in Kirk’s media. The removal was analysed using HPLC, ultra-high performance liquid chromatography (UHPLC) coupled to a QToF mass spectrometer, transmission electron microscopy, and toxicology was assessed using phytotoxicity (Lepidium sativum seeds) and Microtox^® (Allivibrio fisherii) assays. The PHE and BAA degradation rates, in free HMs cultures, reached 78.8% and 70.7%, respectively. However, the addition of HMs considerably affected the BAA degradation rate. The highest degradation rates were associated with the significant production of manganese-peroxidase, lignin peroxidase, and unspecific peroxygenase. The Zn and Cu removal efficacy was considerably higher with live cells than dead cells. Transmission electron microscopy confirmed the involvement of both bioaccumulation and biosorption processes in fungal HM removal. The environmental toxicological assays proved that simultaneous PAH and HM removal was accompanied by detoxification. The metabolites produced during co-treatment were not toxic for plant tissues, and the acute toxicity was reduced. The obtained results indicate that the tested fungi can be applied in the remediation of sites simultaneously contaminated with PAHs and HMs. Full article

With the increasing demand for blue dyes, it is of vital importance to develop a green and efficient biocatalyst to produce indigo. This study constructed a hydrogen peroxide-dependent catalytic system for the direct conversion of indole to indigo using P450BM3 with the assistance of dual-functional small molecules (DFSM). The arrangements of amino acids at 78, 87, and 268 positions influenced the catalytic activity. F87G/T268V mutant gave the highest catalytic activity with k_cat of 1402 min⁻¹ and with a yield of 73%. F87A/T268V mutant was found to produce the indigo product with chemoselectivity as high as 80%. Moreover, F87G/T268A mutant was found to efficiently catalyze indole oxidation with higher activity (k_cat/K_m = 1388 mM⁻¹ min⁻¹) than other enzymes, such as the NADPH-dependent P450BM3 (2.4-fold), the Ngb (32-fold) and the Mb (117-fold). Computer simulation results indicate that the arrangements of amino acid residues in the active site can significantly affect the catalytic activity of the protein. The DFSM-facilitated P450BM3 peroxygenase system provides an alternative, simple approach for a key step in the bioproduction of indigo. Full article

Since 2004, unspecific peroxygenases, in short UPOs (EC. 1.11.2.1), have been explored. UPOs are closing a gap between P450 monooxygenases and chloroperoxidases. These enzymes are highly active biocatalysts for the selective oxyfunctionalisation of C–H, C=C and C-C bonds. UPOs are secreted fungal proteins and Komagataella phaffii (Pichia pastoris) is an ideal host for high throughput screening approaches and UPO production. Heterologous overexpression of 26 new UPOs by K. phaffii was performed in deep well plate cultivation and shake flask cultivation up to 50 mL volume. Enzymes were screened using colorimetric assays with 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (DMP), naphthalene and 5-nitro-1,3-benzodioxole (NBD) as reporter substrates. The PaDa-I (AaeUPO mutant) and HspUPO were used as benchmarks to find interesting new enzymes with complementary activity profiles as well as good producing strains. Herein we show that six UPOs from Psathyrella aberdarensis, Coprinopsis marcescibilis, Aspergillus novoparasiticus, Dendrothele bispora and Aspergillus brasiliensis are particularly active. Full article

Aromatic aldehydes are important aromatic compounds for the flavour and fragrance industry. In this study, a parallel cascade combining aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) and unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO) to convert aromatic primary alcohols into high-value aromatic aldehydes is proposed. Key influencing factors in the process of enzyme cascade catalysis, such as enzyme dosage, pH and temperature, were investigated. The universality of PeAAOx coupled with AaeUPO cascade catalysis for the synthesis of aromatic aldehyde flavour compounds from aromatic primary alcohols was evaluated. In a partially optimised system (comprising 30 μM PeAAOx, 2 μM AaeUPO at pH 7 and 40 °C) up to 84% conversion of 50 mM veratryl alcohol into veratryl aldehyde was achieved in a self-sufficient aerobic reaction. Promising turnover numbers of 2800 and 21,000 for PeAAOx and AaeUPO, respectively, point towards practical applicability. Full article

We recently developed an artificial P450–H₂O₂ system assisted by dual-functional small molecules (DFSMs) to modify the P450BM3 monooxygenase into its peroxygenase mode, which could be widely used for the oxidation of non-native substrates. Aiming to further improve the DFSM-facilitated P450–H₂O₂ system, a series of novel DFSMs having various unnatural amino acid groups was designed and synthesized, based on the co-crystal structure of P450BM3 and a typical DFSM, N-(ω-imidazolyl)-hexanoyl-L-phenylalanine, in this study. The size and hydrophobicity of the amino acid residue in the DFSM drastically affected the catalytic activity (up to 5-fold), stereoselectivity, and regioselectivity of the epoxidation and hydroxylation reactions. Docking simulations illustrated that the differential catalytic ability among the DFSMs is closely related to the binding affinity and the distance between the catalytic group and heme iron. This study not only enriches the DFSM toolbox to provide more options for utilizing the peroxide-shunt pathway of cytochrome P450BM3, but also sheds light on the great potential of the DFSM-driven P450 peroxygenase system in catalytic applications based on DFSM tunability. Full article

Lipid mediators, such as epoxidized or hydroxylated eicosanoids (EETs, HETEs) of arachidonic acid (AA), are important signaling molecules and play diverse roles at different physiological and pathophysiological levels. The EETs and HETEs formed by the cytochrome P450 enzymes are still not fully explored, but show interesting anti-inflammatory properties, which make them attractive as potential therapeutic target or even as therapeutic agents. Conventional methods of chemical synthesis require several steps and complex separation techniques and lead only to low yields. Using the newly discovered unspecific peroxygenase TanUPO from the ascomycetous fungus Truncatella angustata, 90% regioselective conversion of AA to 14,15-EET could be achieved. Selective conversion of AA to 18-HETE, 19-HETE as well as to 11,12-EET and 14,15-EET was also demonstrated with known peroxygenases, i.e., AaeUPO, CraUPO, MroUPO, MweUPO and CglUPO. The metabolites were confirmed by HPLC-ELSD, MS¹ and MS² spectrometry as well as by comparing their analytical data with authentic standards. Protein structure simulations of TanUPO provided insights into its substrate access channel and give an explanation for the selective oxyfunctionalization of AA. The present study expands the scope of UPOs as they can now be used for selective syntheses of AA metabolites that serve as reference material for diagnostics, for structure-function elucidation as well as for therapeutic and pharmacological purposes. Full article

Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in fungi, and catalyze the oxidative degradation of polysaccharides such as cellulose. Despite their name, LPMOs possess a dominant peroxygenase activity that is reflected in high turnover numbers but also causes deactivation. We report on the influence of small molecules and ions on the activity and stability of LPMO during catalysis. Turbidimetric and photometric assays were used to identify LPMO inhibitors and measure their inhibitory effect. Selected inhibitors were employed to study LPMO activity and stability during cellulose depolymerization by HPLC and turbidimetry. It was found that the fungal metabolic products oxalic acid and citric acid strongly reduce LPMO activity, but also protect the enzyme from deactivation. QM calculations showed that the copper atom in the catalytic site could be ligated by bi- or tridentate chelating compounds, which replace two water molecules. MD simulations and QM calculations show that the most likely inhibition pattern is the competition between the inhibitor and reducing agent in the oxidized Cu(II) state. A correlation between the complexation energy and the IC₅₀ values demonstrates that small, bidentate molecules interact strongest with the catalytic site copper and could be used by the fungus as physiological effectors to regulate LPMO activity. Full article

Calcitriol is an active analog of vitamin D3 and has excellent physiological activities in regulating healthy immune function. To synthesize the calcitriol compound, the concept of total synthesis is often adopted, which typically involves multiple steps and results in an overall low yield. Herein, we envisioned an enzymatic approach for the synthesis of calcitriol. Peroxygenase from Agrocybe aegerita (AaeUPO) was used as a catalyst to hydroxylate the C-H bond at the C-25 position of alfacalcidol and yielded the calcitriol in a single step. The enzymatic reaction yielded 80.3% product formation in excellent selectivity, with a turnover number up to 4000. In a semi-preparative scale synthesis, 72% isolated yield was obtained. It was also found that AaeUPO is capable of hydroxylating the C-H bond at the C-1 position of vitamin D3, thereby enabling the calcitriol synthesis directly from vitamin D3. Full article

We reconstructed the molecular phylogeny of heme containing peroxygenases that are known as very versatile biocatalysts. These oxidoreductases capable of mainly oxyfunctionalizations constitute the peroxidase–peroxygenase superfamily. Our representative reconstruction revealed a high diversity but also well conserved sequence motifs within rather short protein molecules. Corresponding genes coding for heme thiolate peroxidases with peroxygenase activity were detected only among various lower eukaryotes. Most of them originate in the kingdom of fungi. However, it seems to be obvious that these htp genes are present not only among fungal Dikarya but they are distributed also in the clades of Mucoromycota and Chytridiomycota with deep ancient evolutionary origins. Moreover, there is also a distinct clade formed mainly by phytopathogenic Stramenopiles where even HTP sequences from Amoebozoa can be found. The phylogenetically older heme peroxygenases are mostly intracellular, but the later evolution gave a preference for secretory proteins mainly among pathogenic fungi. We also analyzed the conservation of typical structural features within various resolved clades of peroxygenases. The presented output of our phylogenetic analysis may be useful in the rational design of specifically modified peroxygenases for various future biotech applications. Full article

The O-demethylation of lignin monomers, which has drawn substantial attention recently, is critical for the formation of phenols from aromatic ethers. The P450BM3 peroxygenase system was recently found to enable the O-demethylation of different aromatic ethers with the assistance of dual-functional small molecules (DFSM), but these prepared mutants only have either moderate O-demethylation activity or moderate selectivity, which hinders their further application. In this study, we improve the system by introducing different amino acids into the active site of P450BM3, and these amino acids with different side chains impacted the catalytic ability of enzymes due to their differences in size, polarity, and hydrophobicity. Among the prepared mutants, the combination of V78A/F87A/T268I/A264G and Im-C6-Phe efficiently catalyzed the O-demethylation of guaiacol (TON = 839) with 100% selectivity. Compared with NADPH-dependent systems, we offer an economical and practical bioconversion avenue. Full article

Vegetable oils are valuable renewable resources for the production of bio-based chemicals and intermediates, including reactive epoxides of industrial interest. Enzymes are an environmentally friendly alternative to chemical catalysis in oxygenation reactions, epoxidation included, with the added advantage of their potential selectivity. The unspecific peroxygenase of Collariella virescens is only available as a recombinant enzyme (rCviUPO), which is produced in Escherichia coli for protein engineering and analytical-scale optimization of plant lipid oxygenation. Engineering the active site of rCviUPO (by substituting one, two, or up to six residues of its access channel by alanines) improved the epoxidation of individual 18-C unsaturated fatty acids and hydrolyzed sunflower oil. The double mutation at the heme channel (F88A/T158A) enhanced epoxidation of polyunsaturated linoleic and α–linolenic acids, with the desired diepoxides representing > 80% of the products (after 99% substrate conversion). More interestingly, process optimization increased (by 100-fold) the hydrolyzate concentration, with up to 85% epoxidation yield, after 1 h of reaction time with the above double variant. Under these conditions, oleic acid monoepoxide and linoleic acid diepoxide are the main products from the sunflower oil hydrolyzate. Full article