Journal articles: 'Peroxygenase activity'

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Zámocký, Marcel, and Jana Harichová. "Evolution of Heme Peroxygenases: Ancient Roots and Later Evolved Branches." Antioxidants 11, no. 5 (May 20, 2022): 1011. http://dx.doi.org/10.3390/antiox11051011.

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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.

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Willot, Tieves, Girhard, Urlacher, Hollmann, and de Gonzalo. "P450BM3-Catalyzed Oxidations Employing Dual Functional Small Molecules." Catalysts 9, no. 7 (June 26, 2019): 567. http://dx.doi.org/10.3390/catal9070567.

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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.

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Kuo, H. H., and A. G. Mauk. "Indole peroxygenase activity of indoleamine 2,3-dioxygenase." Proceedings of the National Academy of Sciences 109, no. 35 (August 13, 2012): 13966–71. http://dx.doi.org/10.1073/pnas.1207191109.

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Hanano, Abdulsamie, Ibrahem Almousally, Mouhnad Shaban, and Elizabeth Blee. "A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection." Applied and Environmental Microbiology 81, no. 18 (June 26, 2015): 6129–44. http://dx.doi.org/10.1128/aem.00867-15.

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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.

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Nguyen, Thi, Soo-Jin Yeom, and Chul-Ho Yun. "Production of a Human Metabolite of Atorvastatin by Bacterial CYP102A1 Peroxygenase." Applied Sciences 11, no. 2 (January 10, 2021): 603. http://dx.doi.org/10.3390/app11020603.

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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.

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Nguyen, Thi Huong Ha, Soo-Jin Yeom, and Chul-Ho Yun. "Production of a Human Metabolite of Atorvastatin by Bacterial CYP102A1 Peroxygenase." Applied Sciences 11, no. 2 (January 10, 2021): 603. http://dx.doi.org/10.3390/app11020603.

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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.

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Molina-Espeja, Patricia, Eva Garcia-Ruiz, David Gonzalez-Perez, René Ullrich, Martin Hofrichter, and Miguel Alcalde. "Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita." Applied and Environmental Microbiology 80, no. 11 (March 28, 2014): 3496–507. http://dx.doi.org/10.1128/aem.00490-14.

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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.

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Molina-Espeja, Patricia, Paloma Santos-Moriano, Eva García-Ruiz, Antonio Ballesteros, Francisco Plou, and Miguel Alcalde. "Structure-Guided Immobilization of an Evolved Unspecific Peroxygenase." International Journal of Molecular Sciences 20, no. 7 (April 2, 2019): 1627. http://dx.doi.org/10.3390/ijms20071627.

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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.

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9

Qin, Xiangquan, Yiping Jiang, Jie Chen, Fuquan Yao, Panxia Zhao, Longyi Jin, and Zhiqi Cong. "Co-Crystal Structure-Guided Optimization of Dual-Functional Small Molecules for Improving the Peroxygenase Activity of Cytochrome P450BM3." International Journal of Molecular Sciences 23, no. 14 (July 18, 2022): 7901. http://dx.doi.org/10.3390/ijms23147901.

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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.

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10

Carballares, Diego, Roberto Morellon-Sterling, Xiaomin Xu, Frank Hollmann, and Roberto Fernandez-Lafuente. "Immobilization of the Peroxygenase from Agrocybe aegerita. The Effect of the Immobilization pH on the Features of an Ionically Exchanged Dimeric Peroxygenase." Catalysts 11, no. 5 (April 28, 2021): 560. http://dx.doi.org/10.3390/catal11050560.

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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.

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11

Sanchez-Sanchez, Lorena, Rosa Roman, and Rafael Vazquez-Duhalt. "Pesticide transformation by a variant of CYPBM3 with improved peroxygenase activity." Pesticide Biochemistry and Physiology 102, no. 2 (February 2012): 169–74. http://dx.doi.org/10.1016/j.pestbp.2011.12.010.

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12

McCombs, Nikolette L., Jennifer D’Antonio, David A. Barrios, Leiah M. Carey, and Reza A. Ghiladi. "Nonmicrobial Nitrophenol Degradation via Peroxygenase Activity of Dehaloperoxidase-Hemoglobin fromAmphitrite ornata." Biochemistry 55, no. 17 (April 22, 2016): 2465–78. http://dx.doi.org/10.1021/acs.biochem.6b00143.

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13

Ramirez-Ramirez, Joaquin, Javier Martin-Diaz, Nina Pastor, Miguel Alcalde, and Marcela Ayala. "Exploring the Role of Phenylalanine Residues in Modulating the Flexibility and Topography of the Active Site in the Peroxygenase Variant PaDa-I." International Journal of Molecular Sciences 21, no. 16 (August 10, 2020): 5734. http://dx.doi.org/10.3390/ijms21165734.

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Unspecific peroxygenases (UPOs) are fungal heme-thiolate enzymes able to catalyze a wide range of oxidation reactions, such as peroxidase-like, catalase-like, haloperoxidase-like, and, most interestingly, cytochrome P450-like. One of the most outstanding properties of these enzymes is the ability to catalyze the oxidation a wide range of organic substrates (both aromatic and aliphatic) through cytochrome P450-like reactions (the so-called peroxygenase activity), which involves the insertion of an oxygen atom from hydrogen peroxide. To catalyze this reaction, the substrate must access a channel connecting the bulk solution to the heme group. The composition, shape, and flexibility of this channel surely modulate the catalytic ability of the enzymes in this family. In order to gain an understanding of the role of the residues comprising the channel, mutants derived from PaDa-I, a laboratory-evolved UPO variant from Agrocybe aegerita, were obtained. The two phenylalanine residues at the surface of the channel, which regulate the traffic towards the heme active site, were mutated by less bulky residues (alanine and leucine). The mutants were experimentally characterized, and computational studies (i.e., molecular dynamics (MD)) were performed. The results suggest that these residues are necessary to reduce the flexibility of the region and maintain the topography of the channel.

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14

Schramm, Marina, Stephanie Friedrich, Kai-Uwe Schmidtke, Jan Kiebist, Paul Panzer, Harald Kellner, René Ullrich, Martin Hofrichter, and Katrin Scheibner. "Cell-Free Protein Synthesis with Fungal Lysates for the Rapid Production of Unspecific Peroxygenases." Antioxidants 11, no. 2 (January 30, 2022): 284. http://dx.doi.org/10.3390/antiox11020284.

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Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal biocatalysts that have attracted considerable interest for application in chemical syntheses due to their ability to selectively incorporate peroxide-oxygen into non-activated hydrocarbons. However, the number of available and characterized UPOs is limited, as it is difficult to produce these enzymes in homologous or hetero-logous expression systems. In the present study, we introduce a third approach for the expression of UPOs: cell-free protein synthesis using lysates from filamentous fungi. Biomass of Neurospora crassa and Aspergillus niger, respectively, was lysed by French press and tested for translational activity with a luciferase reporter enzyme. The upo1 gene from Cyclocybe (Agrocybe) aegerita (encoding the main peroxygenase, AaeUPO) was cell-free expressed with both lysates, reaching activities of up to 105 U L−1 within 24 h (measured with veratryl alcohol as substrate). The cell-free expressed enzyme (cfAaeUPO) was successfully tested in a substrate screening that included prototypical UPO substrates, as well as several pharmaceuticals. The determined activities and catalytic performance were comparable to that of the wild-type enzyme (wtAaeUPO). The results presented here suggest that cell-free expression could become a valuable tool to gain easier access to the immense pool of putative UPO genes and to expand the spectrum of these sought-after biocatalysts.

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Breslmayr, Erik, Peter Poliak, Alen Požgajčić, Roman Schindler, Daniel Kracher, Chris Oostenbrink, and Roland Ludwig. "Inhibition of the Peroxygenase Lytic Polysaccharide Monooxygenase by Carboxylic Acids and Amino Acids." Antioxidants 11, no. 6 (May 31, 2022): 1096. http://dx.doi.org/10.3390/antiox11061096.

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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.

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Hayashi, Takashi, Takaaki Matsuda, and Yoshio Hisaeda. "Enhancement of Peroxygenase Activity of Horse Heart Myoglobin by Modification of Heme-propionate Side Chains." Chemistry Letters 32, no. 6 (June 2003): 496–97. http://dx.doi.org/10.1246/cl.2003.496.

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17

Erman, James E., Heather Kilheeney, Anil K. Bidwai, Caitlan E. Ayala, and Lidia B. Vitello. "Peroxygenase activity of cytochrome c peroxidase and three apolar distal heme pocket mutants: hydroxylation of 1-methoxynaphthalene." BMC Biochemistry 14, no. 1 (2013): 19. http://dx.doi.org/10.1186/1471-2091-14-19.

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Savenkova, Marina I., Jane M. Kuo, and Paul R. Ortiz de Montellano. "Improvement of Peroxygenase Activity by Relocation of a Catalytic Histidine within the Active Site of Horseradish Peroxidase." Biochemistry 37, no. 30 (July 1998): 10828–36. http://dx.doi.org/10.1021/bi9725780.

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19

Mireles, Raul, Joaquin Ramirez-Ramirez, Miguel Alcalde, and Marcela Ayala. "Ether Oxidation by an Evolved Fungal Heme-Peroxygenase: Insights into Substrate Recognition and Reactivity." Journal of Fungi 7, no. 8 (July 28, 2021): 608. http://dx.doi.org/10.3390/jof7080608.

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Ethers can be found in the environment as structural, active or even pollutant molecules, although their degradation is not efficient under environmental conditions. Fungal unspecific heme-peroxygenases (UPO were reported to degrade low-molecular-weight ethers through an H2O2-dependent oxidative cleavage mechanism. Here, we report the oxidation of a series of structurally related aromatic ethers, catalyzed by a laboratory-evolved UPO (PaDa-I) aimed at elucidating the factors influencing this unusual biochemical reaction. Although some of the studied ethers were substrates of the enzyme, they were not efficiently transformed and, as a consequence, secondary reactions (such as the dismutation of H2O2 through catalase-like activity and suicide enzyme inactivation) became significant, affecting the oxidation efficiency. The set of reactions that compete during UPO-catalyzed ether oxidation were identified and quantified, in order to find favorable conditions that promote ether oxidation over the secondary reactions.

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20

Podgorski, Matthew N., Joshua S. Harbort, Joel H. Z. Lee, Giang T. H. Nguyen, John B. Bruning, William A. Donald, Paul V. Bernhardt, Jeffrey R. Harmer, and Stephen G. Bell. "An Altered Heme Environment in an Engineered Cytochrome P450 Enzyme Enables the Switch from Monooxygenase to Peroxygenase Activity." ACS Catalysis 12, no. 3 (January 12, 2022): 1614–25. http://dx.doi.org/10.1021/acscatal.1c05877.

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21

McGuire, Ashlyn H., Leiah M. Carey, Vesna de Serrano, Safaa Dali, and Reza A. Ghiladi. "Peroxidase versus Peroxygenase Activity: Substrate Substituent Effects as Modulators of Enzyme Function in the Multifunctional Catalytic Globin Dehaloperoxidase." Biochemistry 57, no. 30 (June 27, 2018): 4455–68. http://dx.doi.org/10.1021/acs.biochem.8b00540.

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Dezvarei, Shaghayegh, Osami Shoji, Yoshihito Watanabe, and Stephen G. Bell. "The effect of decoy molecules on the activity of the P450Bm3 holoenzyme and a heme domain peroxygenase variant." Catalysis Communications 124 (May 2019): 97–102. http://dx.doi.org/10.1016/j.catcom.2019.03.004.

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Ciaramella, Alberto, Gianluca Catucci, Gianfranco Gilardi, and Giovanna Di Nardo. "Crystal structure of bacterial CYP116B5 heme domain: New insights on class VII P450s structural flexibility and peroxygenase activity." International Journal of Biological Macromolecules 140 (November 2019): 577–87. http://dx.doi.org/10.1016/j.ijbiomac.2019.08.141.

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Cirino, Patrick C., Yi Tang, Katsuyuki Takahashi, David A. Tirrell, and Frances H. Arnold. "Global incorporation of norleucine in place of methionine in cytochrome P450 BM-3 heme domain increases peroxygenase activity." Biotechnology and Bioengineering 83, no. 6 (July 24, 2003): 729–34. http://dx.doi.org/10.1002/bit.10718.

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25

Wei, Xiaoyao, Chun Zhang, Xiaowei Gao, Yanping Gao, Ya Yang, Kai Guo, Xi Du, Lin Pu, and Qin Wang. "Enhanced Activity and Substrate Specificity by Site‐Directed Mutagenesis for the P450 119 Peroxygenase Catalyzed Sulfoxidation of Thioanisole." ChemistryOpen 8, no. 8 (July 2, 2019): 1076–83. http://dx.doi.org/10.1002/open.201900157.

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Li, Maosheng, Hengmin Miao, Yanqing Li, Fang Wang, and Jiakun Xu. "Protein Engineering of an Artificial P450BM3 Peroxygenase System Enables Highly Selective O-Demethylation of Lignin Monomers." Molecules 27, no. 10 (May 13, 2022): 3120. http://dx.doi.org/10.3390/molecules27103120.

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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.

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Hayashi, Takashi, Hideaki Sato, Takashi Matsuo, Takaaki Matsuda, Yutaka Hitomi, and Yoshio Hisaeda. "Enhancement of enzymatic activity for myoglobins by modification of heme-propionate side chains." Journal of Porphyrins and Phthalocyanines 08, no. 03 (March 2004): 255–64. http://dx.doi.org/10.1142/s1088424604000246.

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The modification of myoglobin is an attractive process not only for understanding its molecular mechanism but also for engineering the protein function. The strategy of myoglobin functionalization can be divided into at least two approaches: site-directed mutagenesis and reconstitution with a non-natural prosthetic group. The former method enables us to mainly modulate the physiological function, while the latter has the advantage of introducing a new function on the protein. Particularly, replacement of the native hemin with an artificially created hemin having hydrophobic moieties at the terminal of the heme-propionate side chains serves as an appropriate substrate-binding site near the heme pocket, and consequently enhances the peroxidase and peroxygenase activities for the reconstituted myoglobin. In addition, the incorporation of the synthetic hemin bearing modified heme-propionates into an appropriate apomyoglobin mutant drastically enhances the peroxidase activity. In contrast, to convert myoglobin into a cytochrome P450 enzyme, a flavin moiety as an electron transfer mediator was introduced at the terminal of the heme-propionate side chain. The flavomyoglobin catalyzes the deformylation of 2-phenylpropanal in the presence of NADH under aerobic conditions through the peroxoanion formation from the oxygenated species. In addition, modification of the heme-propionate side chains has an significant influence on regulating the reactivity of the horseradish peroxidase. Furthermore, the heme-propionate side chain can form a metal binding site with a carboxylate residue in the heme pocket. These studies indicate that modification of the heme-propionate side chains can be a new and effective way to engineer functions for the hemoproteins.

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Hlavica, P., I. Golly, M. Lehnerer, and J. Schulze. "Primary aromatic amines: their N-oxidative bioactivation." Human & Experimental Toxicology 16, no. 8 (August 1997): 441–48. http://dx.doi.org/10.1177/096032719701600805.

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There exists a diversity of pathways in mammalian cells serving to activate primary aromatic amines. 1 N-Oxidative mixed-function turnover usually involves participation of the cytochrome P450 superfamily, while catalysis by the flavin-containing monooxy genases is restricted to a few amines capable of forming imine tautomers. Surprisingly, haemoglobin metabo lizes cytotoxic and carcinogenic arylamines via a monooxygenase-like mechanism, but peroxygenase activity is also operative. 2 In extrahepatic tissues that exhibit only a low level of monooxygenases, peroxidative transformations, as are brought about by prostaglandin H synthase, myeloperoxidase or lactoperoxidase, predominate in amine activation. Non-mammalian peroxidases fre quently used as model systems include horseradish peroxidase and chloroperoxidase. 3 Non-enzymatic, light-induced conversion of aromatic amines to free radical or N-oxy products proceeds either via direct photolysis of the nitrogenous com pounds or through attack by lipid-derived reactive intermediates generated during irradiation. 4 The interplay of the various tissue-specific processes of arylamine activation serves to explain differences in susceptibility toward the biological actions of primary aromatic amines.

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Díaz-Quintana, Antonio, Gonzalo Pérez-Mejías, Alejandra Guerra-Castellano, Miguel A. De la Rosa, and Irene Díaz-Moreno. "Wheel and Deal in the Mitochondrial Inner Membranes: The Tale of Cytochrome c and Cardiolipin." Oxidative Medicine and Cellular Longevity 2020 (April 22, 2020): 1–20. http://dx.doi.org/10.1155/2020/6813405.

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Cardiolipin oxidation and degradation by different factors under severe cell stress serve as a trigger for genetically encoded cell death programs. In this context, the interplay between cardiolipin and another mitochondrial factor—cytochrome c—is a key process in the early stages of apoptosis, and it is a matter of intense research. Cytochrome c interacts with lipid membranes by electrostatic interactions, hydrogen bonds, and hydrophobic effects. Experimental conditions (including pH, lipid composition, and post-translational modifications) determine which specific amino acid residues are involved in the interaction and influence the heme iron coordination state. In fact, up to four binding sites (A, C, N, and L), driven by different interactions, have been reported. Nevertheless, key aspects of the mechanism for cardiolipin oxidation by the hemeprotein are well established. First, cytochrome c acts as a pseudoperoxidase, a process orchestrated by tyrosine residues which are crucial for peroxygenase activity and sensitivity towards oxidation caused by protein self-degradation. Second, flexibility of two weakest folding units of the hemeprotein correlates with its peroxidase activity and the stability of the iron coordination sphere. Third, the diversity of the mode of interaction parallels a broad diversity in the specific reaction pathway. Thus, current knowledge has already enabled the design of novel drugs designed to successfully inhibit cardiolipin oxidation.

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Hangasky, John A., Anthony T. Iavarone, and Michael A. Marletta. "Reactivity of O2 versus H2O2 with polysaccharide monooxygenases." Proceedings of the National Academy of Sciences 115, no. 19 (April 23, 2018): 4915–20. http://dx.doi.org/10.1073/pnas.1801153115.

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Enzymatic conversion of polysaccharides into lower-molecular-weight, soluble oligosaccharides is dependent on the action of hydrolytic and oxidative enzymes. Polysaccharide monooxygenases (PMOs) use an oxidative mechanism to break the glycosidic bond of polymeric carbohydrates, thereby disrupting the crystalline packing and creating new chain ends for hydrolases to depolymerize and degrade recalcitrant polysaccharides. PMOs contain a mononuclear Cu(II) center that is directly involved in C–H bond hydroxylation. Molecular oxygen was the accepted cosubstrate utilized by this family of enzymes until a recent report indicated reactivity was dependent on H2O2. Reported here is a detailed analysis of PMO reactivity with H2O2 and O2, in conjunction with high-resolution MS measurements. The cosubstrate utilized by the enzyme is dependent on the assay conditions. PMOs will directly reduce O2 in the coupled hydroxylation of substrate (monooxygenase activity) and will also utilize H2O2 (peroxygenase activity) produced from the uncoupled reduction of O2. Both cosubstrates require Cu reduction to Cu(I), but the reaction with H2O2 leads to nonspecific oxidation of the polysaccharide that is consistent with the generation of a hydroxyl radical-based mechanism in Fenton-like chemistry, while the O2 reaction leads to regioselective substrate oxidation using an enzyme-bound Cu/O2 reactive intermediate. Moreover, H2O2 does not influence the ability of secretome from Neurospora crassa to degrade Avicel, providing evidence that molecular oxygen is a physiologically relevant cosubstrate for PMOs.

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Shen, Yue, Qing-Li Jia, Ming-Zhe Liu, Zhuo-Wei Li, Li-Li Wang, Cui-Zhu Zhao, Zhi-Xi Li, and Meng Zhang. "Genome-wide characterization and phylogenetic and expression analyses of the caleosin gene family in soybean, common bean and barrel medic." Archives of Biological Sciences 68, no. 3 (2016): 575–85. http://dx.doi.org/10.2298/abs150916048s.

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Caleosin are a class of calcium-binding proteins embedded in the phospholipid monolayer of lipid droplets. In addition to maintaining thestructure of lipid droplets, caleosin proteins areinvolved in dormancy and lipid signaling, and areassociatedwith the stress response via their histidine-dependent peroxygenase activity. To date, caleosins have been studied in Arabidopsis thaliana. However, little is known about these genes in legumes,including the most cultivated oilseed crop, soybean. In this paper,20 caleosin genes in soybean, common bean and barrel medic werestudied. Among these, 13 caleosin genes, including 3 in Glycine max, 5 in Phaseolus vulgarisand 5 in Medicago truncatula, are identified for the first time. The structures, characteristics and evolution of the 20 caleosin proteins are analyzed. Expansion patterns show that tandem duplication was the main reason for the caleosin family expansion in the legume. Expression profiles indicate that L-caleosin in soybean and common bean are more important than H-caleosin, which is just the opposite in Arabidopsis thaliana. GmaCLO2, PvuCLO1, PvuCLO3and MtrCLO3may play important roles, while GmaCLO6, GmaCLO10and MtrCLO4may lose their function in the examined tissues. In addition, according to the results of cis-element analyses, we propose potential functions for the more important caleosin genes in leguminous plants. Our work provides helpful information for further evolution and function analyses of the caleosin gene family in soybean, common bean and barrel medic.

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Matsuo, Takashi, and Takashi Hayashi. "Electron transfer and oxidase activities in reconstituted hemoproteins with chemically modified cofactors." Journal of Porphyrins and Phthalocyanines 13, no. 10 (October 2009): 1082–89. http://dx.doi.org/10.1142/s1088424609001340.

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Protoheme IX is a typical iron porphyrin cofactor, showing a variety of reactivities in many hemoproteins under the reaction environments provided by protein matrices. Chemical modification of the protoheme cofactor is expected to be a versatile strategy to design hemoproteins possessing unique functions. This review focuses on the conversion of a hemoprotein, mainly myoglobin (an oxygen-storage hemoprotein), into a protein having different functions from the original ones by replacement of the protoheme cofactor with synthetic cofactors. The myoglobin having anionic patches pended to the heme propionates effectively binds electron-accepting proteins or small cationic organic molecules on the protein surface, resulting in enhanced efficiency of the photoinduced electron transfers from the myoglobin to these electron acceptors. Furthermore, the peroxidase and peroxygenase activities are also enhanced due to the facile substrate accesses. The attachment of the chemically active moiety such as flavin at the heme terminal is also important to give P450-like function to the native myoglobin. The employment of a structural isomer of porphyrin as an artificial cofactor gives rise to remarkably high dioxygen affinity and peroxidase activity in myoglobin, and allows us to easily detect high-valent species of the porphyrin isomer in HRP. These examples provide a clear insight into hemoprotein modifications based on synthetic chemistry as well as genetic amino acid mutations.

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Lappe, Alessa, Nina Jankowski, Annemie Albrecht, and Katja Koschorreck. "Characterization of a thermotolerant aryl-alcohol oxidase from Moesziomyces antarcticus oxidizing 5-hydroxymethyl-2-furancarboxylic acid." Applied Microbiology and Biotechnology 105, no. 21-22 (October 13, 2021): 8313–27. http://dx.doi.org/10.1007/s00253-021-11557-8.

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Abstract The development of enzymatic processes for the environmentally friendly production of 2,5-furandicarboxylic acid (FDCA), a renewable precursor for bioplastics, from 5-hydroxymethylfurfural (HMF) has gained increasing attention over the last years. Aryl-alcohol oxidases (AAOs) catalyze the oxidation of HMF to 5-formyl-2-furancarboxylic acid (FFCA) through 2,5-diformylfuran (DFF) and have thus been applied in enzymatic reaction cascades for the production of FDCA. AAOs are flavoproteins that oxidize a broad range of benzylic and aliphatic allylic primary alcohols to the corresponding aldehydes, and in some cases further to acids, while reducing molecular oxygen to hydrogen peroxide. These promising biocatalysts can also be used for the synthesis of flavors, fragrances, and chemical building blocks, but their industrial applicability suffers from low production yield in natural and heterologous hosts. Here we report on heterologous expression of a new aryl-alcohol oxidase, MaAAO, from Moesziomyces antarcticus at high yields in the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). Fed-batch fermentation of recombinant P. pastoris yielded around 750 mg of active enzyme per liter of culture. Purified MaAAO was highly stable at pH 2–9 and exhibited high thermal stability with almost 95% residual activity after 48 h at 57.5 °C. MaAAO accepts a broad range of benzylic primary alcohols, aliphatic allylic alcohols, and furan derivatives like HMF as substrates and some oxidation products thereof like piperonal or perillaldehyde serve as building blocks for pharmaceuticals or show health-promoting effects. Besides this, MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) to FFCA, which has not been shown for any other AAO so far. Combining MaAAO with an unspecific peroxygenase oxidizing HMFCA to FFCA in one pot resulted in complete conversion of HMF to FDCA within 144 h. MaAAO is thus a promising biocatalyst for the production of precursors for bioplastics and bioactive compounds. Key points • MaAAO from M. antarcticus was expressed in P. pastoris at 750 mg/l. • MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). • Complete conversion of HMF to 2,5-furandicarboxylic acid by combining MaAAO and UPO.

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Anh, Dau Hung, René Ullrich, Dirk Benndorf, Aleś Svatoś, Alexander Muck, and Martin Hofrichter. "The Coprophilous Mushroom Coprinus radians Secretes a Haloperoxidase That Catalyzes Aromatic Peroxygenation." Applied and Environmental Microbiology 73, no. 17 (June 29, 2007): 5477–85. http://dx.doi.org/10.1128/aem.00026-07.

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ABSTRACT Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent K m values for ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 μM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.

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Ebner, Katharina, Lukas J. Pfeifenberger, Claudia Rinnofner, Veronika Schusterbauer, Anton Glieder, and Margit Winkler. "Discovery and Heterologous Expression of Unspecific Peroxygenases." Catalysts 13, no. 1 (January 16, 2023): 206. http://dx.doi.org/10.3390/catal13010206.

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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.

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Chen, Zhifeng, Jie Chen, Nana Ma, Haifeng Zhou, and Zhiqi Cong. "Selective hydroxylation of naphthalene using the H2O2-dependent engineered P450BM3 driven by dual-functional small molecules." Journal of Porphyrins and Phthalocyanines 22, no. 09n10 (August 21, 2018): 831–36. http://dx.doi.org/10.1142/s108842461850061x.

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We herein report the H2O2-dependent selective hydroxylation of naphthalene catalyzed by engineered P450BM3 with the assistance of dual-functional small molecules (DFSMs). The mutation at position 268 significantly improved the hydroxylation activity of P450BM3, which is quite different from those engineered P450BM3 peroxygenases and NADPH-dependent P450BM3 mutants previously reported, implicating the unique role of the residue at position 268. This study provides a potential approach to develop the practical hydroxylation biocatalyst of P450s for aromatic hydrocarbons using the DFSM-facilitated P450BM3-H2O2 system.

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Pickl, Mathias, Sara Kurakin, Fabián G. Cantú Reinhard, Philipp Schmid, Alexander Pöcheim, Christoph K. Winkler, Wolfgang Kroutil, Sam P. de Visser, and Kurt Faber. "Mechanistic Studies of Fatty Acid Activation by CYP152 Peroxygenases Reveal Unexpected Desaturase Activity." ACS Catalysis 9, no. 1 (December 6, 2018): 565–77. http://dx.doi.org/10.1021/acscatal.8b03733.

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Karich, Alexander, Katrin Scheibner, René Ullrich, and Martin Hofrichter. "Exploring the catalase activity of unspecific peroxygenases and the mechanism of peroxide-dependent heme destruction." Journal of Molecular Catalysis B: Enzymatic 134 (December 2016): 238–46. http://dx.doi.org/10.1016/j.molcatb.2016.10.014.

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Cho, Won-Il, and Myong-Soo Chung. "Bacillus spores: a review of their properties and inactivation processing technologies." Food Science and Biotechnology 29, no. 11 (October 6, 2020): 1447–61. http://dx.doi.org/10.1007/s10068-020-00809-4.

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Abstract Many factors determine the resistance properties of a Bacillus spore to heat, chemical and physical processing, including thick proteinaceous coats, peptidoglycan cortex and low water content, high levels of dipicolinic acid (DPA), and divalent cations in the spore core. Recently, attention has been focused on non-thermal inactivation methods based on high pressure, ultrasonic, high voltage electric fields and cold plasmas for inactivating Bacillus spores associated with deterioration in quality and safety. The important chemical sporicides are glutaraldehyde, chorine-releasing agents, peroxygens, and ethylene oxide. Some food-grade antimicrobial agents exhibit sporostatic and sporicidal activities, such as protamine, polylysine, sodium lactate, essential oils. Surfactants with hydrophilic and hydrophobic properties have been reported to have inactivation activity against spores. The combined treatment of physical and chemical treatment such as heating, UHP (ultra high pressure), PEF (pulsed electric field), UV (ultraviolet), IPL (intense pulsed light) and natural antimicrobial agents can act synergistically and effectively to kill Bacillus spores in the food industry.

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Cantú Reinhard, Fabián G., Yen-Ting Lin, Agnieszka Stańczak, and Sam P. de Visser. "Bioengineering of Cytochrome P450 OleTJE: How Does Substrate Positioning Affect the Product Distributions?" Molecules 25, no. 11 (June 9, 2020): 2675. http://dx.doi.org/10.3390/molecules25112675.

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The cytochromes P450 are versatile enzymes found in all forms of life. Most P450s use dioxygen on a heme center to activate substrates, but one class of P450s utilizes hydrogen peroxide instead. Within the class of P450 peroxygenases, the P450 OleTJE isozyme binds fatty acid substrates and converts them into a range of products through the α-hydroxylation, β-hydroxylation and decarboxylation of the substrate. The latter produces hydrocarbon products and hence can be used as biofuels. The origin of these product distributions is unclear, and, as such, we decided to investigate substrate positioning in the active site and find out what the effect is on the chemoselectivity of the reaction. In this work we present a detailed computational study on the wild-type and engineered structures of P450 OleTJE using a combination of density functional theory and quantum mechanics/molecular mechanics methods. We initially explore the wild-type structure with a variety of methods and models and show that various substrate activation transition states are close in energy and hence small perturbations as through the protein may affect product distributions. We then engineered the protein by generating an in silico model of the double mutant Asn242Arg/Arg245Asn that moves the position of an active site Arg residue in the substrate-binding pocket that is known to form a salt-bridge with the substrate. The substrate activation by the iron(IV)-oxo heme cation radical species (Compound I) was again studied using quantum mechanics/molecular mechanics (QM/MM) methods. Dramatic differences in reactivity patterns, barrier heights and structure are seen, which shows the importance of correct substrate positioning in the protein and the effect of the second-coordination sphere on the selectivity and activity of enzymes.

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Gjorgjeska, Biljana, and Dino Karpicarov. "MECHANISM OF ACTION AND CHARACTERISTICS OF CERTAIN ANTISEPTICS AND DISINFECTANTS IN CORRELATION WITH THEIR ACTIVITY ON SELECTED MICROORGANISMS." Knowledge International Journal 28, no. 2 (December 10, 2018): 423–28. http://dx.doi.org/10.35120/kij2802423g.

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Antiseptics and disinfectants represent a large group of compounds such as: alcohols, aldehydes, acid and base compounds, anilides, biguanides, diamidines, halogen release agents, heavy metals and their compounds, peroxygens, phenols, bis–phenols, halophenols, quaternary ammonium compounds and volatile compounds for sterilization. Both antiseptics and disinfectants are labeled as biocides which are compounds that have the ability to destroy microorganisms or prevent their growth, development and reproduction. Usually, when referring to biocides that inhibit growth, other terms may be more specific, such as “–static” and when referring to biocides that kill the target microorganism the term “–cidal” is often used. These chemical compounds have different effects depending on the concentration in which they are used. The main difference between antiseptics and disinfectants is the place of application. As such, antiseptics remove microorganisms (bacteria, fungi, viruses, parasites that have varying degree of pathogenicity and virulence) from living tissues while disinfectants remove the same type of microorganisms from variety of objects and equipment, or to remove pathogens from the immediate environment. The action of antiseptics and disinfectants is due to mutual reaction with the cell surface of the microorganisms, followed by their penetration into the cells and the influence on a certain target area. As a result of that, antiseptics and disinfectants are an integral part of the practices for controlling infections and preventing the occurrence of intra–hospital infections. One of the biggest problems facing modern medicine is the occurrence of the intra–hospital (inpatient, nosocomial) infections. These infections can be defined as localized or generalized infections caused by microorganisms acquired during hospitalization. More specifically, an intra–hospital infection is one for which there is no evidence that the infection was present or incubating at the time of a hospital admission. In fact, these infections can result from inappropriate use of antiseptics and disinfectants. To be used in hospital conditions, antiseptics and disinfectants must meet several criteria: easy to use; non–volatile; not harmful to equipment, staff or patients; free from unpleasant smells and effective within a relatively short time.The goals of this study are to present the most common microorganisms that cause the occurrence of intra–hospital infections; to present the characteristics and mechanisms of action of the most frequently used antiseptics and disinfectants in hospital conditions; to give guidance as to which antiseptic or disinfectant would be most suitable for use against the microorganism which occurs in the function of the causative agent of the intra–hospital infection. The establishment of such an approach is crucial because it is necessary to know which antiseptic or disinfectant has the greatest activity against the microorganism which is the cause of the intra–hospital (nosocomial) infection. As a result of that, the incidence of intra–hospital infections will be minimized.

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Kont, Riin, Bastien Bissaro, Vincent G. H. Eijsink, and Priit Väljamäe. "Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs)." Nature Communications 11, no. 1 (November 13, 2020). http://dx.doi.org/10.1038/s41467-020-19561-8.

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AbstractLytic polysaccharide monooxygenases (LPMOs) are widely distributed in Nature, where they catalyze the hydroxylation of glycosidic bonds in polysaccharides. Despite the importance of LPMOs in the global carbon cycle and in industrial biomass conversion, the catalytic properties of these monocopper enzymes remain enigmatic. Strikingly, there is a remarkable lack of kinetic data, likely due to a multitude of experimental challenges related to the insoluble nature of LPMO substrates, like cellulose and chitin, and to the occurrence of multiple side reactions. Here, we employed competition between well characterized reference enzymes and LPMOs for the H2O2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation. LPMOs of both bacterial and fungal origin showed high peroxygenase efficiencies, with kcat/KmH2O2 values in the order of 105–106 M−1 s−1. Besides providing crucial insight into the cellulolytic peroxygenase reaction, these results show that LPMOs belonging to multiple families and active on multiple substrates are true peroxygenases.

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Martin-Diaz, Javier, Carmen Paret, Eva García-Ruiz, Patricia Molina-Espeja, and Miguel Alcalde. "Shuffling the Neutral Drift of Unspecific Peroxygenase inSaccharomyces cerevisiae." Applied and Environmental Microbiology 84, no. 15 (May 18, 2018). http://dx.doi.org/10.1128/aem.00808-18.

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ABSTRACTUnspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO inSaccharomyces cerevisiae, so that neutral mutations were simply added and recombinedin vivo. With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded active UPO variants were generated. After only eight rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with changes in peroxidative and peroxygenative activities, kinetic thermostability, and enhanced tolerance to organic solvents. With an average of 4.6 substitutions introduced per clone, neutral mutations covered approximately 10% of the protein sequence. Accordingly, this study opens new avenues for UPO design by bringing together neutral genetic drift and DNA recombinationin vivo.IMPORTANCEFungal peroxygenases resemble the peroxide shunt pathway of cytochrome P450 monoxygenases, performing selective oxyfunctionalizations of unactivated C-H bonds in a broad range of organic compounds. In this study, we combined neutral genetic drift andin vivoDNA shuffling to generate highly functional peroxygenase mutant libraries. The panel of neutrally evolved peroxygenases showed different activity profiles for peroxygenative substrates and improved stability with respect to temperature and the presence of organic cosolvents, making the enzymes valuable blueprints for emerging evolution campaigns. This association of DNA recombination and neutral drift is paving the way for future work in peroxygenase engineering and, from a more general perspective, to any other enzyme system heterologously expressed inS. cerevisiae.

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Freakley, Simon J., Svenja Kochius, Jacqueline van Marwijk, Caryn Fenner, Richard J. Lewis, Kai Baldenius, Sarel S. Marais, et al. "A chemo-enzymatic oxidation cascade to activate C–H bonds with in situ generated H2O2." Nature Communications 10, no. 1 (September 13, 2019). http://dx.doi.org/10.1038/s41467-019-12120-w.

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Abstract Continuous low-level supply or in situ generation of hydrogen peroxide (H2O2) is essential for the stability of unspecific peroxygenases, which are deemed ideal biocatalysts for the selective activation of C–H bonds. To envisage potential large scale applications of combined catalytic systems the reactions need to be simple, efficient and produce minimal by-products. We show that gold-palladium nanoparticles supported on TiO2 or carbon have sufficient activity at ambient temperature and pressure to generate H2O2 from H2 and O2 and supply the oxidant to the engineered unspecific heme-thiolate peroxygenase PaDa-I. This tandem catalyst combination facilitates efficient oxidation of a range of C-H bonds to hydroxylated products in one reaction vessel with only water as a by-product under conditions that could be easily scaled.

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Harlington, Alix C., Keith E. Shearwin, Stephen G. Bell, and Fiona Whelan. "Efficient O-demethylation of lignin monoaromatics using the peroxygenase activity of cytochrome P450 enzymes." Chemical Communications, 2022. http://dx.doi.org/10.1039/d2cc04698a.

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Leone, Linda, Daniele D'Alonzo, Véronique Balland, Gerardo Zambrano, Marco Chino, Flavia Nastri, Ornella Maglio, Vincenzo Pavone, and Angela Lombardi. "Mn-Mimochrome VI*a: An Artificial Metalloenzyme With Peroxygenase Activity." Frontiers in Chemistry 6 (December 4, 2018). http://dx.doi.org/10.3389/fchem.2018.00590.

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Molina-Espeja, Patricia, Alejandro Beltran-Nogal, Maria Alejandra Alfuzzi, Victor Guallar, and Miguel Alcalde. "Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita." Frontiers in Bioengineering and Biotechnology 9 (September 14, 2021). http://dx.doi.org/10.3389/fbioe.2021.741282.

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Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel.

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Molina-Espeja, Patricia, Alejandro Beltran-Nogal, Maria Alejandra Alfuzzi, Victor Guallar, and Miguel Alcalde. "Corrigendum: Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase From Agrocybe aegerita." Frontiers in Bioengineering and Biotechnology 9 (October 5, 2021). http://dx.doi.org/10.3389/fbioe.2021.778727.

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Molina-Espeja, Patricia, Alejandro Beltran-Nogal, Maria Alejandra Alfuzzi, Victor Guallar, and Miguel Alcalde. "Corrigendum: Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase From Agrocybe aegerita." Frontiers in Bioengineering and Biotechnology 9 (October 5, 2021). http://dx.doi.org/10.3389/fbioe.2021.778727.

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Mate, Diana M., Miguel A. Palomino, Patricia Molina-Espeja, Javier Martin-Diaz, and Miguel Alcalde. "Modification of the peroxygenative:peroxidative activity ratio in the unspecific peroxygenase fromAgrocybe aegeritaby structure-guided evolution." Protein Engineering Design and Selection, January 1, 2017. http://dx.doi.org/10.1093/protein/gzw073.

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Journal articles on the topic 'Peroxygenase activity'

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