Добірка наукової літератури з теми "Peroxygenase activity"

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.

A set of dual functional small molecules (DFSMs) containing different amino acids has been synthesized and employed together with three different variants of the cytochrome P450 monooxygenase P450BM3 from Bacillus megaterium in H2O2-dependent oxidation reactions. These DFSMs enhance P450BM3 activity with hydrogen peroxide as an oxidant, converting these enzymes into formal peroxygenases. This system has been employed for the catalytic epoxidation of styrene and in the sulfoxidation of thioanisole. Various P450BM3 variants have been evaluated in terms of activity and selectivity of the peroxygenase reactions.

ABSTRACTCaleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein inAspergillus flavusthat is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway,aflRandaflD, were downregulated in the strains in whichA. flavusPXG(AfPXG) was silenced, leading to reduced aflatoxin B1 productionin vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in whichAfPXGwas silenced.PXG-deficientA. flavusstrains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease.

Atorvastatin is a widely used statin drug that prevents cardiovascular disease and treats hyperlipidemia. The major metabolites in humans are 2-OH and 4-OH atorvastatin, which are active metabolites known to show highly inhibiting effects on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Producing the hydroxylated metabolites by biocatalysts using enzymes and whole-cell biotransformation is more desirable than chemical synthesis. It is more eco-friendly and can increase the yield of desired products. In this study, we have found an enzymatic strategy of P450 enzymes for highly efficient synthesis of the 4-OH atorvastatin, which is an expensive commercial product, by using bacterial CYP102A1 peroxygenase activity with hydrogen peroxide without NADPH. We obtained a set of CYP102A1 mutants with high catalytic activity toward atorvastatin using enzyme library generation, high-throughput screening of highly active mutants, and enzymatic characterization of the mutants. In the hydrogen peroxide supported reactions, a mutant, with nine changed amino acid residues compared to a wild-type among tested mutants, showed the highest catalytic activity of atorvastatin 4-hydroxylation (1.8 min−1). This result shows that CYP102A1 can catalyze atorvastatin 4-hydroxylation by peroxide-dependent oxidation with high catalytic activity. The advantages of CYP102A1 peroxygenase activity over NADPH-supported monooxygenase activity are discussed. Taken together, we suggest that the P450 peroxygenase activity can be used to produce drugs’ metabolites for further studies of their efficacy and safety.

Atorvastatin is a widely used statin drug that prevents cardiovascular disease and treats hyperlipidemia. The major metabolites in humans are 2-OH and 4-OH atorvastatin, which are active metabolites known to show highly inhibiting effects on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Producing the hydroxylated metabolites by biocatalysts using enzymes and whole-cell biotransformation is more desirable than chemical synthesis. It is more eco-friendly and can increase the yield of desired products. In this study, we have found an enzymatic strategy of P450 enzymes for highly efficient synthesis of the 4-OH atorvastatin, which is an expensive commercial product, by using bacterial CYP102A1 peroxygenase activity with hydrogen peroxide without NADPH. We obtained a set of CYP102A1 mutants with high catalytic activity toward atorvastatin using enzyme library generation, high-throughput screening of highly active mutants, and enzymatic characterization of the mutants. In the hydrogen peroxide supported reactions, a mutant, with nine changed amino acid residues compared to a wild-type among tested mutants, showed the highest catalytic activity of atorvastatin 4-hydroxylation (1.8 min−1). This result shows that CYP102A1 can catalyze atorvastatin 4-hydroxylation by peroxide-dependent oxidation with high catalytic activity. The advantages of CYP102A1 peroxygenase activity over NADPH-supported monooxygenase activity are discussed. Taken together, we suggest that the P450 peroxygenase activity can be used to produce drugs’ metabolites for further studies of their efficacy and safety.

ABSTRACTUnspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected theAgrocybe aegeritaUPO1-encoding gene to directed evolution inSaccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using anad hocdual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in thekcat/Kmvalue for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme’s activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.

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.

This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47 °C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. The enzyme was not immobilized on glyoxyl-agarose supports, while it was immobilized albeit in inactive form on vinyl-sulfone-activated supports. rAaeUPO immobilization on glutaraldehyde pre-activated supports gave almost quantitative immobilization yield and retained some activity, but the biocatalyst was very unstable. Its immobilization via anion exchange on PEI supports also produced good immobilization yields, but the rAaeUPO stability dropped. However, using aminated agarose, the enzyme retained stability and activity. The stability of the immobilized enzyme strongly depended on the immobilization pH, being much less stable when rAaeUPO was adsorbed at pH 9.0 than when it was immobilized at pH 7.0 or pH 5.0 (residual activity was almost 0 for the former and 80% for the other preparations), presenting stability very similar to that of the free enzyme. This is a very clear example of how the immobilization pH greatly affects the final biocatalyst performance.

The ability of the cytochrome P450 heme monooxygenases to catalyze difficult oxidation reactions, often with high specificity and selectivity, makes them attractive for numerous biotechnological applications. However they are generally limited by low turnover rates and low stability, and their minimum requirements for catalysis include a cofactor as source of electrons (NAD(P)H), partner proteins for electron transfer, and dioxygen. Some P450s are capable of supporting low levels of peroxygenase activity, in which a peroxide is utilized to drive catalysis via a "shunt" pathway. This mechanism for substrate oxidation, although inefficient and not generally utilized in nature, simplifies P450 catalysis by eliminating the need for NAD(P)H.

Our goal was to engineer an efficient P450 peroxygenase which utilizes hydrogen peroxide (H₂O₂). Directed evolution is a powerful enzyme engineering methodology which mimics nature's algorithm for evolution. Enzyme libraries are generated via DNA mutagenesis or recombination techniques, and variants with improved function are isolated using an appropriate screen or selection. Using this strategy, in combination with site-directed mutagenesis, we have created P450 BM-3 heme domain variants with more than 100-fold improved H₂O₂-driven hydroxylation activity compared to wild-type, showing both an improved k_cat as well as a lower K_m for H₂O₂. Thermostability was also improved by directed evolution.

We have engineered a cell-free, biomimetic hydroxylase that requires only H₂O₂ to exploit the hydroxylating power of P450 BM-3. Peroxide-mediated inactivation as a result of heme destruction remains a major obstacle and presents an important enzyme engineering challenge. This research has broadened the potential applications of P450 biocatalysis by exploiting the versatility of heme-containing proteins.

The cytochrome P450 heme-thiolate enzymes catalyse a multitude of oxidation reactions and, in humans, carry out drug metabolism. P450s perform hydroxylation, epoxidation, N-, O- and Sdealkylation, sulfoxidation, alkyne oxidation and aldehyde oxidation of organic molecules (and many other reactions). These reactions are predominantly performed by the reactive intermediate Compound I, but other intermediates in the catalytic cycle may mediate some types of reactions. It would be appealing to exploit these enzymes as environmentally benign catalysts in the synthesis of fine chemicals. Their widespread use in industrial synthesis is, however, impractical given the high cost of the required cofactor NAD(P)H, but engineering P450s to instead use cheap H₂O₂ would overcome this problem. CYP199A4 is a soluble bacterial P450 enzyme from Rhodopseudomonas palustris HaA2 that favours 4-methoxybenzoic acid and other para-substituted benzoic acids as substrates. It tightly binds these substrates and the para-substituent is rapidly oxidised. This enzyme has been used as a model system to study the mechanism of P450-catalysed reactions. While 4-methoxybenzoic acid is oxidatively demethylated at a rate of 1220 μM (μM-P450)⁻¹ min⁻¹, CYP199A4 displays no detectable activity towards the meta isomer, 3-methoxybenzoic acid. In vitro reactions were performed with a range of other meta-substituted benzoic acids (3-methylthio-, 3-methylamino-, 3- formyl-, 3-methyl-, 3-isopropyl-, 3-tert-butyl- and 3-ethoxy-benzoic acid) to assess whether CYP199A4 had activity towards these substrates. These meta-substituted substrates, except for 3- tert-butylbenzoic acid, were all metabolised by CYP199A4, but with low activity compared to the corresponding para isomers. Compared to the para isomers, the meta isomers had lower binding affinity and induced smaller type I spin-state shifts to high-spin. To rationalise CYP199A4’s preference for para- over meta-substituted benzoic acid substrates and to investigate the requirements for efficient monooxygenase activity, crystal structures were solved of CYP199A4 in complex with 3-methoxy-, 3-methylamino-, 3-methylthio-, and 3- and 4-methyl-benzoic acid. These structures revealed that the heme-bound water ligand to the heme is retained when these substrates bind (water occupancy 21-90%) and is hydrogen-bonded to the heteroatom (N, S, O) of the substrate. The corresponding para isomers displace the iron-bound water. 3-Ethoxybenzoic acid, which has a bulkier meta-substituent, shifted the spin-state to 85% high-spin and in the crystal structure the iron-bound water was removed. The meta-substituent of each substrate is held in close proximity to the iron. 3-Methoxybenzoic acid is positioned near the iron but is not oxidised. This was attributed to the fact that the C-H bonds are oriented away from the heme, whereas those of 4-methoxybenzoic acid are ideally oriented for H-atom abstraction by Cpd I. These results emphasise that close proximity of the methyl carbon to the heme iron does not guarantee that hydroxylation will occur if the C-H bonds are not oriented appropriately for abstraction, and subtle modification of the substrate’s position relative to the heme can abolish catalytic activity. X-ray crystallography, CW and HYSCORE EPR and other experiments were performed to elucidate the binding modes of 4-pyridin-2-yl-, 4-pyridin-3-yl- and 4-imidazol-1-yl-benzoic acid in the CYP199A4 active site. These heterocyclic aromatic compounds are not metabolised and induce substantially different type II UV-Vis spectra. 4-Pyridin-3-yl- and 4-imidazol-1-yl-benzoic acid redshifted the Soret band from 419 to 424 nm. They induced ‘normal’ type II spectra, characterised by a less intense α-band than β-band and an increase in δ-band intensity. The UV-Vis spectra of ferrous CYP199A4 in complex with these ligands indicated that 4-pyridin-3-ylbenzoic acid was directly ligated to the heme iron via the pyridine nitrogen, but the Fe-N bond between the iron and 4-imidazol-1-ylbenzoic acid was ruptured upon heme reduction. 4-Pyridin-2-ylbenzoic acid induced a smaller Soret band red-shift (to 422 nm) when added to the ferric enzyme. It produced an ‘abnormal’ type II spectrum, with no decrease in the α-band intensity. 4-Pyridin-2-ylbenzoic acid also induced a smaller Soret band trough in the difference spectrum than 4-pyridin-3-ylbenzoic acid. HYSCORE EPR and X-ray crystallography revealed that 4-pyridin-3-yl- and 4-imidazol-1-ylbenzoic acid were directly ligated to the ferric heme iron, but 4-pyridin-2-ylbenzoic acid was hydrogen-bonded to the heme-bound water. This study revealed that optical spectroscopy can distinguish between water-bridged and directly bound nitrogen donor ligands. 4-Pyridin-3-yl- and 4-imidazol-1-yl-benzoic acid-bound CYP199A4 were both reduced to the ferrous form by ferredoxin, ferredoxin reductase and NADH. On the other hand, binding of 4-pyridin-2-ylbenzoic acid to CYP199A4 lowered the reduction potential and prevented heme reduction by even the powerful reductant dithionite. This implies that water-bridged nitrogen ligands may in some instances be more effective P450 inhibitors than those that bind directly to the iron. The T252E mutant of CYP199A4 was produced and characterised. This variant was no longer able to operate using NADH but was a more efficient peroxygenase (H₂O₂-utilising enzyme) than the wild-type (WT) enzyme. EPR indicated that the sixth axial ligand to the heme was a mixture of hydroxide and water. Crystal structures showed that this aqua/hydroxo ligand was tightly bound due to strong interactions with the carboxylate of E252. The axial aqua/hydroxo ligand was not displaced by substrates, even sterically bulky substrates, explaining the lack of substrate-induced spin-state shifts and the exceedingly slow rate of electron transfer from the ferredoxin to the P450. Because this ligand is not displaced by substrates, the active species could potentially be generated via light-driven oxidation of the water-bound ferric resting state to Compound I. Type II nitrogen ligands were also unable to displace the aqua/hydroxo ligand. 4-Pyridin-3-ylbenzoic acid, when added to the T252E mutant, induced an ‘abnormal’ type II spectrum, confirming that optical spectroscopy can distinguish between water-bridged and directly bound type II ligands. X-ray crystallography revealed that the T252 → E mutation only subtly altered the orientation of substrates in the CYP199A4 active site. In the absence of substrate, the heme signal of the T252E mutant was rapidly bleached by 50 mM H₂O₂. When substrate was present, the T252E variant remained catalytically active for several hours. The T252E variant was able to perform a range of reactions using H₂O₂ (O-dealkylation, hydroxylation/desaturation, epoxidation, sulfoxidation, alkyne oxidation and aldehyde oxidation). When the surrogate oxygen donor tert-butyl hydroperoxide was substituted for H₂O₂, the T252E mutant had negligible activity. t-BuOOH is presumably too bulky to access the heme iron in this P450. WT CYP199A4 and the T252A and D251N mutants also catalysed these reactions using H₂O₂ but afforded less product over a 4-hour period than the T252E mutant. H₂O₂- and NADH-driven epoxidation of 4-vinylbenzoic acid catalysed by WT and mutant CYP199A4 proceeded with high enantioselectivity, yielding almost exclusively the (S)-enantiomer. In NADH-supported reactions, WT CYP199A4 catalysed O-demethylation of 4- methoxybenzoic acid more efficiently than sulfoxidation of 4-methylthiobenzoic acid. In H₂O₂- driven reactions, the T252E variant had higher activity towards sulfoxidation compared to Odemethylation, hydroxylation, epoxidation, aldehyde oxidation and alkyne oxidation. This may indicate the involvement of a second oxidant in sulfoxidation (e.g. the FeIII–H₂O₂ species), allowing sulfoxidation to occur in the absence of Compound I as proposed by Shaik.
Thesis (MPhil) -- University of Adelaide, School of Physical Sciences, 2020