Heme peroxygenases exhibit remarkable catalytic versatility in facilitating a wide array of oxidative reactions under mild conditions,eliminating the need for coenzymes and intricate electron transport systems.This un...Heme peroxygenases exhibit remarkable catalytic versatility in facilitating a wide array of oxidative reactions under mild conditions,eliminating the need for coenzymes and intricate electron transport systems.This unique character underscores their essentiality and potential as promising tools in synthetic biology.Recent advancements in enzyme engineering have significantly enhanced the catalytic performance of both natural and artificial peroxygenases.Extensive engineering efforts have been directed towards unspecific peroxygenases and fatty acid peroxygenases,aiming to expand their substrate specificities,and enhance reaction selectivities,as well as increase enzyme stability.Furthermore,innovative strategies such as dual-functional small molecule-assisted systems and H_(2)O_(2) tunnel engineering have been harnessed to transform P450 monooxygenases into highly efficient peroxygenases,capable of catalyzing reactions with a variety of unnatural substrates.This review consolidates the latest progress in the engineered and artificial heme peroxygenases,emphasizing their catalytic performances as potent biocatalysts for sustainable organic synthesis.展开更多
Unspecific peroxygenases(UPOs, EC 1.11.2.1) is a kind of thioheme enzyme capable of catalyzing various oxidations of inert C–H bonds using H_(2)O_(2) as an oxygen donor without cofactors. However, the enhancement of ...Unspecific peroxygenases(UPOs, EC 1.11.2.1) is a kind of thioheme enzyme capable of catalyzing various oxidations of inert C–H bonds using H_(2)O_(2) as an oxygen donor without cofactors. However, the enhancement of the H_(2)O_(2) tolerance of UPOs is always challenging. In this study, the A161C mutant of r Dca UPO,which originates from Daldinia caldariorum, was found to be highly H_(2)O_(2)-resistant. Compared with the wild type, the mutant r Dca UPO-A161C showed a 10-h prolonged half-life and a 64% improved enzyme activity when incubated in 10 mmol/L H_(2)O_(2). The crystal structure analysis at 1.47 A showed that r Dca UPOA161C exhibited 10 α-helixes(cyan) and a series of ordered rings, forming a single asymmetric spherical structure. The two conserved domains near heme formed an active site with the catalytic PCP and EHD regions(Glu86, His87, Asp88 residues). The H_(2)O_(2) tolerance of r Dca UPO-A161C was preliminarily explored by comparing its structure with the wild type. Notably, r Dca UPO-A161C showed significantly higher catalytic efficiency than the wild type for the production of hydroxyl fatty acids. This study is anticipated to provide an insight into the structure-function relationship and expand potential applications of UPOs.展开更多
Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterolog...Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterologous expression has hindered their practical application.Here,a novel UPO from Marasmius fiardii PR910(MfiUPO)was identified and heterologously expressed in Pichia pastoris.By employing a two-copy expression cassette,the protein titer reached 1.18 g L−1 in a 5 L bioreactor,marking the highest record.The glycoprotein rMfiUPO exhib-ited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation,epoxidation and alcohol oxidation.Moreover,the peroxidative activity was enhanced by 150%after exposure to 50%(v/v)acetone for 40 h.A semipreparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2%isolated yield with 95%purity.With its high expression level,rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution,thereby meeting the necessary criteria for practical application.展开更多
Musk macrolides are a kind of rare flavors structurally featuring 14-16 membered rings in the flavour and fragrance industry.While various chemical synthesis routes for musk macrolides exist,there is a growing interes...Musk macrolides are a kind of rare flavors structurally featuring 14-16 membered rings in the flavour and fragrance industry.While various chemical synthesis routes for musk macrolides exist,there is a growing interest in biocatalytic approaches.In this study,a new biocatalytic approach is proposed for the synthesis of musk macrolides from fatty acids using unspecific peroxygenases(UPOs)in combination with lipase.Specifically,the UPO from Marasmius rotula(MroUPO)was utilized as an oxidation catalyst together with the choline oxidase from Arthrobacter nicotinate(AnChOx)for the in situ generation of H_(2)O_(2).The enzymatic cascade process involved the hydroxylation of fatty acids using MroUPO as a catalyst to prepare terminal hydroxylated fatty acids.Under the optimized conditions,14-hydroxy tetradecanoic acid,15-hydroxy pentadecanoic acid and 16-hydroxy hexadecanoic acid were obtained at concentrations of 11.1,18.2 and17.1 mg/L in a 50-mL reaction system,respectively.These hydroxylated fatty acids were then separated and esterified by lipase Novozymes 435 to produce the desired macrolides.Variousω-hydroxy fatty acids were successfully converted into macrolides,with the concentrations of cyclotetradecanolide,cyclopentadecanolide and cyclohexadecanolide reaching 11,29 and 23 mg/L,respectively,in a 10-mL reaction system.This new biocatalytic approach represents a promising method for synthesizing valuable natural flavour compounds.展开更多
Indigoids,a class of bis-indoles,have long been applied in dyeing,food,and pharmaceutical industries.Recently,interest in these‘old’molecules has been renewed in the field of organic semiconductors as functional bui...Indigoids,a class of bis-indoles,have long been applied in dyeing,food,and pharmaceutical industries.Recently,interest in these‘old’molecules has been renewed in the field of organic semiconductors as functional building blocks for organic electronics due to their excellent chemical and physical properties.However,these indigo derivatives are difficult to access through chemical synthesis.In this study,we engineer cytochrome P450 BM3 from an NADPH-dependent monooxygenase to peroxygenases through directed evolution.A select number of P450 BM3 variants are used for the selective oxidation of indole derivatives to form different indigoid pigments with a spectrum of colors.Among the prepared indigoid organic photocatalysts,a majority of indigoids demonstrate a reduced band gap than indigo due to the increased light capture and improved charge separation,making them promising candidates for the development of new organic electronic devices.Thus,we present a useful enzymatic approach with broad substrate scope and cost-effectiveness by using low-cost H2O2 as a cofactor for the preparation of diversified indigoids,offering versatility in designing and manufacturing new dyestuff and electronic/sensor components.展开更多
The cardiovascular effects of vitamin C (VitC) could be mediated by epoxyeicosatrienoic acids (EETs). We aimed to study the mechanism of VitC-dependent microsomal formation of cis- and trans-EETs and the regulation of...The cardiovascular effects of vitamin C (VitC) could be mediated by epoxyeicosatrienoic acids (EETs). We aimed to study the mechanism of VitC-dependent microsomal formation of cis- and trans-EETs and the regulation of EET levels in rat isolated perfused kidneys and in vivo. VitC biphasically stimulated rat kidney microsomal cis- and trans-EET formation in a ratio of 1:2, involving the participation of lipid hydroperoxides (LOOHs), Fe2+ , and cytochrome P450 (CYP). Levels of LOOHs correlated with microsomal EET production. LOOH stimulation of CYP isoforms resulted in preferred trans- over cis-EET formation from arachidonic acid and was associated with the cleavage of LOOHs, which indicated a CYP peroxy-genase activity. EETs contributed to VitC-induced vasodilator responses in rat isolated perfused kidneys. VitC (1 mg/ml) given in the drinking water for 9 days doubled rat urinary EET excretion, increased plasma levels of EETs, mostly trans-EETs, by 40%, and reduced plasma levels of 20-hydroxyeicosatetraenoic acid. Depletion of VitC in brain cortex and kidney tissues by more than 20- and 50-fold, respectively, in gulonolactone oxidase-knockout mice was associated with mild increases in tissue EETs. These data suggest that LOOHs are a determinant factor for EET formation in vivo in which VitC exerts a key regulatory effect. VitC-activated CYP peroxygenase activity may represent a CYP interaction with lipoxygenases and cyclooxygenases to mediate the cardiovascular effects of VitC via formation of EETs.展开更多
It is demonstrated that (3Z)-nonenal (NON) and (3Z)-hexenal (HEX) are oxidized in a cascade by lipoxygenase (LOX) and hydroperoxide peroxygenase (HP peroxygenase) into (2E)-4-hydroxy-2- nonenal (HNE) and (2E)-4-hydrox...It is demonstrated that (3Z)-nonenal (NON) and (3Z)-hexenal (HEX) are oxidized in a cascade by lipoxygenase (LOX) and hydroperoxide peroxygenase (HP peroxygenase) into (2E)-4-hydroxy-2- nonenal (HNE) and (2E)-4-hydroxy-2-hexenal (HHE), respectively. In turn, HNE inactivates LOX terminating the cascade. The hydroxy-alkenals produced serve to inhibit plant pathogens, which initiated the cascade. In addition to LOX, other unknown oxygenases may be involved in the cascade.展开更多
Unspecific peroxygenases exhibit high activity for the selective oxyfunctionalization of inert C(sp3)–H bonds using only H_(2)O_(2) as a clean oxidant,while also exhibiting sensitivity to H_(2)O_(2) concentration.CdS...Unspecific peroxygenases exhibit high activity for the selective oxyfunctionalization of inert C(sp3)–H bonds using only H_(2)O_(2) as a clean oxidant,while also exhibiting sensitivity to H_(2)O_(2) concentration.CdS-based semiconductors are promising for the photosynthesis of H_(2)O_(2) owing to their adequately negative potential for oxygen reduction reaction via a proton-coupled electron transfer process,however,they suffer from fast H_(2)O_(2) decomposition on the surface of pristine CdS.Therefore,[Cp*Rh(bpy)H_(2)O]2+,a highly selective proton-coupled electron transfer catalyst,was anchored onto a supramolecular polymer-grafted CdS nanoflower to construct an efficient integrated photocatalyst for generating H_(2)O_(2),mitigating the surface issue of pristine CdS,increasing light absorption,accelerating photonic carrier separation,and enhancing oxygen reduction reaction selectivity to H_(2)O_(2).This photocatalyst promoted the light driven H_(2)O_(2) generation rate up to 1345μmol·L^(-1)·g^(-1)·h^(-1),which was 2.4 times that of pristine CdS.The constructed heterojunction photocatalyst could supply H_(2)O_(2) in situ for nonspecific peroxygenases to catalyze the C–H oxyfunctionalization of ethylbenzene,achieving a yield of 81%and an ee value of 99%under optimum conditions.A wide range of substrates were converted to the corresponding chiral alcohols using this photo-enzyme catalytic system,achieving the corresponding chiral alcohols in good yield(51%–88%)and excellent enantioselectivity(90%–99%ee).展开更多
文摘Heme peroxygenases exhibit remarkable catalytic versatility in facilitating a wide array of oxidative reactions under mild conditions,eliminating the need for coenzymes and intricate electron transport systems.This unique character underscores their essentiality and potential as promising tools in synthetic biology.Recent advancements in enzyme engineering have significantly enhanced the catalytic performance of both natural and artificial peroxygenases.Extensive engineering efforts have been directed towards unspecific peroxygenases and fatty acid peroxygenases,aiming to expand their substrate specificities,and enhance reaction selectivities,as well as increase enzyme stability.Furthermore,innovative strategies such as dual-functional small molecule-assisted systems and H_(2)O_(2) tunnel engineering have been harnessed to transform P450 monooxygenases into highly efficient peroxygenases,capable of catalyzing reactions with a variety of unnatural substrates.This review consolidates the latest progress in the engineered and artificial heme peroxygenases,emphasizing their catalytic performances as potent biocatalysts for sustainable organic synthesis.
基金supported by the National Natural Science Foundation of China (No.32001633)the Key Program of Natural Science Foundation of China (No.31930084)Guangzhou Science and technology planning project (No.202102020370)。
文摘Unspecific peroxygenases(UPOs, EC 1.11.2.1) is a kind of thioheme enzyme capable of catalyzing various oxidations of inert C–H bonds using H_(2)O_(2) as an oxygen donor without cofactors. However, the enhancement of the H_(2)O_(2) tolerance of UPOs is always challenging. In this study, the A161C mutant of r Dca UPO,which originates from Daldinia caldariorum, was found to be highly H_(2)O_(2)-resistant. Compared with the wild type, the mutant r Dca UPO-A161C showed a 10-h prolonged half-life and a 64% improved enzyme activity when incubated in 10 mmol/L H_(2)O_(2). The crystal structure analysis at 1.47 A showed that r Dca UPOA161C exhibited 10 α-helixes(cyan) and a series of ordered rings, forming a single asymmetric spherical structure. The two conserved domains near heme formed an active site with the catalytic PCP and EHD regions(Glu86, His87, Asp88 residues). The H_(2)O_(2) tolerance of r Dca UPO-A161C was preliminarily explored by comparing its structure with the wild type. Notably, r Dca UPO-A161C showed significantly higher catalytic efficiency than the wild type for the production of hydroxyl fatty acids. This study is anticipated to provide an insight into the structure-function relationship and expand potential applications of UPOs.
基金funded by the National Key Research and Development Program of China(2021YFC2102700)was Supported by the Key Science and Technology Innovation Project of Hubei Province(2021BAD001)as well as the Research Program of State Key Laboratory of Biocatalysis and Enzyme Engineering。
文摘Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterologous expression has hindered their practical application.Here,a novel UPO from Marasmius fiardii PR910(MfiUPO)was identified and heterologously expressed in Pichia pastoris.By employing a two-copy expression cassette,the protein titer reached 1.18 g L−1 in a 5 L bioreactor,marking the highest record.The glycoprotein rMfiUPO exhib-ited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation,epoxidation and alcohol oxidation.Moreover,the peroxidative activity was enhanced by 150%after exposure to 50%(v/v)acetone for 40 h.A semipreparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2%isolated yield with 95%purity.With its high expression level,rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution,thereby meeting the necessary criteria for practical application.
基金supported by the Key Realm R&D Program of Guangdong Province(2022B0202050003)National Natural Science Foundation of China(31930084,32001633)。
文摘Musk macrolides are a kind of rare flavors structurally featuring 14-16 membered rings in the flavour and fragrance industry.While various chemical synthesis routes for musk macrolides exist,there is a growing interest in biocatalytic approaches.In this study,a new biocatalytic approach is proposed for the synthesis of musk macrolides from fatty acids using unspecific peroxygenases(UPOs)in combination with lipase.Specifically,the UPO from Marasmius rotula(MroUPO)was utilized as an oxidation catalyst together with the choline oxidase from Arthrobacter nicotinate(AnChOx)for the in situ generation of H_(2)O_(2).The enzymatic cascade process involved the hydroxylation of fatty acids using MroUPO as a catalyst to prepare terminal hydroxylated fatty acids.Under the optimized conditions,14-hydroxy tetradecanoic acid,15-hydroxy pentadecanoic acid and 16-hydroxy hexadecanoic acid were obtained at concentrations of 11.1,18.2 and17.1 mg/L in a 50-mL reaction system,respectively.These hydroxylated fatty acids were then separated and esterified by lipase Novozymes 435 to produce the desired macrolides.Variousω-hydroxy fatty acids were successfully converted into macrolides,with the concentrations of cyclotetradecanolide,cyclopentadecanolide and cyclohexadecanolide reaching 11,29 and 23 mg/L,respectively,in a 10-mL reaction system.This new biocatalytic approach represents a promising method for synthesizing valuable natural flavour compounds.
基金the National Key Research and Development Program of China(2019YFA0905100)the National Natural Science Foundation of China(32025001,32071266 and 32170088)+1 种基金the Shandong Provincial Natural Science Foundation(ZR2019ZD20)the State Key Laboratory of Microbial Technology Open Projects Fund(Project NO.M2022-01).
文摘Indigoids,a class of bis-indoles,have long been applied in dyeing,food,and pharmaceutical industries.Recently,interest in these‘old’molecules has been renewed in the field of organic semiconductors as functional building blocks for organic electronics due to their excellent chemical and physical properties.However,these indigo derivatives are difficult to access through chemical synthesis.In this study,we engineer cytochrome P450 BM3 from an NADPH-dependent monooxygenase to peroxygenases through directed evolution.A select number of P450 BM3 variants are used for the selective oxidation of indole derivatives to form different indigoid pigments with a spectrum of colors.Among the prepared indigoid organic photocatalysts,a majority of indigoids demonstrate a reduced band gap than indigo due to the increased light capture and improved charge separation,making them promising candidates for the development of new organic electronic devices.Thus,we present a useful enzymatic approach with broad substrate scope and cost-effectiveness by using low-cost H2O2 as a cofactor for the preparation of diversified indigoids,offering versatility in designing and manufacturing new dyestuff and electronic/sensor components.
文摘The cardiovascular effects of vitamin C (VitC) could be mediated by epoxyeicosatrienoic acids (EETs). We aimed to study the mechanism of VitC-dependent microsomal formation of cis- and trans-EETs and the regulation of EET levels in rat isolated perfused kidneys and in vivo. VitC biphasically stimulated rat kidney microsomal cis- and trans-EET formation in a ratio of 1:2, involving the participation of lipid hydroperoxides (LOOHs), Fe2+ , and cytochrome P450 (CYP). Levels of LOOHs correlated with microsomal EET production. LOOH stimulation of CYP isoforms resulted in preferred trans- over cis-EET formation from arachidonic acid and was associated with the cleavage of LOOHs, which indicated a CYP peroxy-genase activity. EETs contributed to VitC-induced vasodilator responses in rat isolated perfused kidneys. VitC (1 mg/ml) given in the drinking water for 9 days doubled rat urinary EET excretion, increased plasma levels of EETs, mostly trans-EETs, by 40%, and reduced plasma levels of 20-hydroxyeicosatetraenoic acid. Depletion of VitC in brain cortex and kidney tissues by more than 20- and 50-fold, respectively, in gulonolactone oxidase-knockout mice was associated with mild increases in tissue EETs. These data suggest that LOOHs are a determinant factor for EET formation in vivo in which VitC exerts a key regulatory effect. VitC-activated CYP peroxygenase activity may represent a CYP interaction with lipoxygenases and cyclooxygenases to mediate the cardiovascular effects of VitC via formation of EETs.
文摘It is demonstrated that (3Z)-nonenal (NON) and (3Z)-hexenal (HEX) are oxidized in a cascade by lipoxygenase (LOX) and hydroperoxide peroxygenase (HP peroxygenase) into (2E)-4-hydroxy-2- nonenal (HNE) and (2E)-4-hydroxy-2-hexenal (HHE), respectively. In turn, HNE inactivates LOX terminating the cascade. The hydroxy-alkenals produced serve to inhibit plant pathogens, which initiated the cascade. In addition to LOX, other unknown oxygenases may be involved in the cascade.
基金supported by the National Natural Science Foundation of China(Grant No.22378096)the Natural Science Foundation of Hebei Province(Grant No.B2023202014)+1 种基金the Science Technology Research Project of Higher Education of Hebei Province(Grant Nos.QN2021045,and QN2023207)the Tianjin Science and Technology Project(Grant No.22KPHDRC00260).
文摘Unspecific peroxygenases exhibit high activity for the selective oxyfunctionalization of inert C(sp3)–H bonds using only H_(2)O_(2) as a clean oxidant,while also exhibiting sensitivity to H_(2)O_(2) concentration.CdS-based semiconductors are promising for the photosynthesis of H_(2)O_(2) owing to their adequately negative potential for oxygen reduction reaction via a proton-coupled electron transfer process,however,they suffer from fast H_(2)O_(2) decomposition on the surface of pristine CdS.Therefore,[Cp*Rh(bpy)H_(2)O]2+,a highly selective proton-coupled electron transfer catalyst,was anchored onto a supramolecular polymer-grafted CdS nanoflower to construct an efficient integrated photocatalyst for generating H_(2)O_(2),mitigating the surface issue of pristine CdS,increasing light absorption,accelerating photonic carrier separation,and enhancing oxygen reduction reaction selectivity to H_(2)O_(2).This photocatalyst promoted the light driven H_(2)O_(2) generation rate up to 1345μmol·L^(-1)·g^(-1)·h^(-1),which was 2.4 times that of pristine CdS.The constructed heterojunction photocatalyst could supply H_(2)O_(2) in situ for nonspecific peroxygenases to catalyze the C–H oxyfunctionalization of ethylbenzene,achieving a yield of 81%and an ee value of 99%under optimum conditions.A wide range of substrates were converted to the corresponding chiral alcohols using this photo-enzyme catalytic system,achieving the corresponding chiral alcohols in good yield(51%–88%)and excellent enantioselectivity(90%–99%ee).
