N-substituted furfurylamines(FAs)are valuable precursors for producing pharmacologically active compounds and polymers.However,enzymatic synthesis of the type of chemicals is still in its infancy.Here we report an imi...N-substituted furfurylamines(FAs)are valuable precursors for producing pharmacologically active compounds and polymers.However,enzymatic synthesis of the type of chemicals is still in its infancy.Here we report an imine reductase from Streptomyces albidoflavus(SaIRED)for the reductive amination of biobased furans.A simple,fast and interference-resistant high-throughput screening(HTS)method was developed,based on the coloration reaction of carbonyl compounds with 2,4-dinitrophenylhydrazine.The reductive amination activity of IREDs can be directly indicated by a colorimetric assay.With the reductive amination of furfural with allylamine as the model reaction,SaIRED with the activity of 4.8 U mg^(-1) was subjected to three rounds of protein engineering and screening by this HTS method,affording a high-activity tri-variant I127V/D241A/A242T(named M3,20.2 U mg^(-1)).The variant M3 showed broad substrate scope,and enabled efficient reductive amination of biobased furans with a variety of amines including small aliphatic amines and sterically hindered amines,giving the target FAs in yields up to>99%.In addition,other variants were identified for preparative-scale synthesis of commercially interesting amines such as N-2-(methylsulfonyl)ethyl-FA by the screen method,with isolated yields up to 87%and turnover numbers up to 9700 for enzyme.Gram-scale synthesis of N-allyl-FA,a valuable building block and potential polymer monomer,was implemented at 0.25 mol L^(-1) substrate loading by a whole-cell catalyst incorporating variant M3,with 4.7 g L^(-1) h^(-1) space-time yield and 91%isolated yield.展开更多
Aldo-keto reductases(AKRs)are a superfamily of enzymes that play crucial roles in various cellular processes,including the metabolism of xenobiotics,steroids,and carbohydrates.A growing body of evidence has unveiled t...Aldo-keto reductases(AKRs)are a superfamily of enzymes that play crucial roles in various cellular processes,including the metabolism of xenobiotics,steroids,and carbohydrates.A growing body of evidence has unveiled the involvement of AKRs in the development and progression of various cancers.AKRs are aberrantly expressed in a wide range of malignant tumors.Dysregulated expression of AKRs enables the acquisition of hallmark traits of cancer by activating oncogenic signaling pathways and contributing to chemoresistance.AKRs have emerged as promising oncotherapeutic targets given their pivotal role in cancer development and progression.Inhibition of aldose reductase(AR),either alone or in combination with chemotherapeutic drugs,has evolved as a pragmatic therapeutic option for cancer.Several classes of synthetic aldo-keto reductase(AKR)inhibitors have been developed as potential anticancer agents,some of which have shown promise in clinical trials.Many AKR inhibitors from natural sources also exhibit anticancer effects.Small molecule inhibitors targeting specific AKR isoforms have shown promise in preclinical studies.These inhibitors disrupt the activation of oncogenic signaling by modulating transcription factors and kinases and sensitizing cancer cells to chemotherapy.In this review,we discuss the physiological functions of human AKRs,the aberrant expression of AKRs in malignancies,the involvement of AKRs in the acquisition of cancer hallmarks,and the role of AKRs in oncogenic signaling,and drug resistance.Finally,the potential of aldose reductase inhibitors(ARIs)as anticancer drugs is summarized.展开更多
Optically pure(R)-γ-and(R)-δ-lactones can be prepared by intramolecular cyclization of chiral hydroxy acids/esters reduced asymmetrically from γ-and δ-keto acids/esters using Saccharomyces cerevisiae(S.cerevisiae)...Optically pure(R)-γ-and(R)-δ-lactones can be prepared by intramolecular cyclization of chiral hydroxy acids/esters reduced asymmetrically from γ-and δ-keto acids/esters using Saccharomyces cerevisiae(S.cerevisiae) as a whole-cell biocatalyst.However,some of the enzymes catalyzing these reactions in S.cerevisiae are still unknown up to date.In this report,two carbonyl reductases,OdCRl and OdCR2,were successfully discovered,and cloned from S.cerevisiae using a genome-mining approach,and overexpressed in Escherichia coli(E.coli).Compared with OdCR1,OdCR2 can reduce 4-oxodecanoic acid and 5-oxodecanoic acid asymmetrically with higher stereoselectivity,generating(R)-γ-decalactone(99% ee) and(R)-δ-decalactone(98% ee) in 85% and 92%yields,respectively.This is the first report of native enzymes from S.cerevisiae for the enzymatic synthesis of chiral γ-and δ-lactones which is of wide uses in food and cosmetic industries.展开更多
Introduction One of the major root alkaloids of the Indian medicinal plant Rauvolfia serpenlina Benth. ex Kurz is named ajmaline. The enzymatic biosynthesis of this alkaloid has been studied for a long time by our gr...Introduction One of the major root alkaloids of the Indian medicinal plant Rauvolfia serpenlina Benth. ex Kurz is named ajmaline. The enzymatic biosynthesis of this alkaloid has been studied for a long time by our group As a result, a biosynthetic pathway has been established, in which about 10 enzymes participate, several of them belonging to the group of NADPH-dependent reductases. Started with the biosynthetic precursors tryptamine and secologanin,展开更多
The thermostability of three sulfur oxygenase reductases （SORs） was investigated from thermoacidophilic achaea Acidianus tengchongensis （SORAT） and Sulfolobus tokodaii （SORsT） as well as the moderately thermophi...The thermostability of three sulfur oxygenase reductases （SORs） was investigated from thermoacidophilic achaea Acidianus tengchongensis （SORAT） and Sulfolobus tokodaii （SORsT） as well as the moderately thermophilic bacterium Acidithiobacillus sp. SM-1 （SORsB）. The optimal temperatures for catalyzing sulfur oxidation were 80 ℃ （SORAT）, 85 ℃ （SORsT）, and 70 ℃ （SORsB）, respectively. The half-lives of the three SORs at their optimal catalytic conditions were 100 min （SORAT）, 58 min （SORsT）, and 37 min （SORsB）. In order to reveal the structural basis of the thermostability of these SORs, three-dimensional structural models of them were generated by homology modeling using the previously reported high-resolution X-ray structure of SORAA （from Acidianus ambivalens） as a template. The results suggest that thermostability was dependent on： （a） high number of the charged amino acid glutamic acid and the flexible amino acid proline, （b） low number of the therraolabile amino acid glutamine, （c） increased number of ion pairs, （d） decreased ratio of hydrophobie accessible solvent surface area （ASA） to charged ASA, and （e） increased volumes of the cavity. The number of cavities and the number of hydrogen bonds did not signifieantly affect the thermostability of SORs, whereas the cavity volumes increased as the thermal stability increased.展开更多
Mining and tailings deposition can cause serious heavy metal(loids)pollution to the surrounding soil environment.Soil microorganisms adapt their metabolism to such conditions,driving alterations in soil function.This ...Mining and tailings deposition can cause serious heavy metal(loids)pollution to the surrounding soil environment.Soil microorganisms adapt their metabolism to such conditions,driving alterations in soil function.This study aims to elucidate the response patterns of nitrogen-cycling microorganisms under long-term heavy metal(loids)exposure.The results showed that the diversity and abundance of nitrogen-cyclingmicroorganisms showed negative feedback to heavy metal(loids)concentrations.Denitrifying microorganisms were shown to be the dominant microorganisms with over 60%of relative abundance and a complex community structure including 27 phyla.Further,the key bacterial species in the denitrification process were calculated using a random forest model,where the top three key species(Pseudomonas stutzei,Sphingobium japonicum and Leifsonia rubra)were found to play a prominent role in nitrite reduction.Functional gene analysis and qPCR revealed that nirK,which is involved in nitrite reduction,significantly accumulated in the most metal-rich soil with the increase of absolute abundance of 63.86%.The experimental results confirmed that the activity of nitrite reductase(Nir)encoded by nirK in the soil was increased at high concentrations of heavy metal(loids).Partial least squares-path model identified three potential modes of nitrite reduction processes being stimulated by heavy metal(loids),the most prominent of which contributed to enhanced nirK abundance and soil Nir activity through positive stimulation of key species.The results provide new insights and preliminary evidence on the stimulation of nitrite reduction processes by heavy metal(loids).展开更多
Surgical intervention is currently the primary treatment for hepatolithiasis;how-ever,some patients still experience residual stones and high recurrence rates after surgery.Cholesterol metabolism seems to play an impo...Surgical intervention is currently the primary treatment for hepatolithiasis;how-ever,some patients still experience residual stones and high recurrence rates after surgery.Cholesterol metabolism seems to play an important role in hepatoli-thiasis pathogenesis.A high cholesterol diet is one of the significant reasons for the increasing incidence of hepatolithiasis.Therefore,regular diet and appropriate medical intervention are crucial measures to prevent hepatolithiasis and reduce recurrence rate after surgery.Reducing dietary cholesterol and drugs that increase cholesterol stone solubility are key therapeutic approaches in treating hepato-lithiasis.This article discusses the cholesterol metabolic pathways related to the pathogenesis of hepatolithiasis,as well as food intake and targeted therapeutic drugs.展开更多
Ethyl(R)-4-chloro-3-hydroxybutyrate((R)-CHBE),as a chiral intermediate,is widely used in the synthesis of various chiral drugs.In this study,we screened two aldo–keto reductases(LP-AKRs)from the probiotic Lactobacill...Ethyl(R)-4-chloro-3-hydroxybutyrate((R)-CHBE),as a chiral intermediate,is widely used in the synthesis of various chiral drugs.In this study,we screened two aldo–keto reductases(LP-AKRs)from the probiotic Lactobacillus plantarum DSM20174,both with a molecular weight of approximately 31 kDa.Both enzymes could reduce 4-chloroacetoacetic acid ethyl ester(COBE)to produce(R)-CHBE with an enantioselectivity value of 99%.When determining the kinetic parameter,the K_(m),K_(cat),and V_(max)of LP-AKR5 and LP-AKR9 were 9.5 mM,7.6 U/mg,3.96 s^(-1)and 8.7 mM,8.59 U/mg,4.47 s^(-1),respectively.Both LP-AKR5 and LP-AKR9 had an optimal reaction pH of 6 and could maintain a high level of stability at pH 6,allowing them to perform well in an acidic environment.LP-AKR5 and LP-AKR9 had optimal reaction temperatures of 30℃and 40℃,respectively.Metal ions had minimal influence on LP-AKR5 and LP-AKR9 enzyme activities.This series of enzymatic properties showed that LP-AKR5 and LP-AKR9 mined from Lactobacillus plantarum DSM20174 could asym-metrically catalyze the synthesis of(R)-CHBE under weakly acidic circumstances,which could maintain product stability and provide a good foundation for industrial production.展开更多
Microbial selenite(Se(Ⅳ)) reduction to elemental selenium nanoparticles(SeNPs) and chromate(Cr(Ⅵ)) remediation constitute effective detoxification processes. The strain Bacillus wiedmannii ES2-45 can efficiently red...Microbial selenite(Se(Ⅳ)) reduction to elemental selenium nanoparticles(SeNPs) and chromate(Cr(Ⅵ)) remediation constitute effective detoxification processes. The strain Bacillus wiedmannii ES2-45 can efficiently reduce 5 mmol/L Se(Ⅳ) into SeNPs over 14 h and 1 mmol/L Cr(Ⅵ) within 36 h. Proteomic analysis and qRT-PCR revealed that reductases, including dithiol oxidoreductase(DsbA), Fe-S biosynthesis domain-containing protein(Fes),and aldose-6-phosphate reductase(Aldo), and bacillithiol(BSH) were involved in Se(Ⅳ) reduction. Heterologous expression further confirmed the Se(Ⅳ)-reducing function of three genes and enhanced the Se(Ⅳ) and Cr(Ⅵ) resistance ability. Moreover, the difference between Se(Ⅳ) treatments and controls both intra-and extra-cellular BSH concentration per biomass indicated that BSH contributes to Se(Ⅳ) reduction. Selenium-fortified rice was obtained by foliar spray of SeNPs synthesized by the strain ES2-45. To the best of our knowledge, DsbA and Aldo reductase were firstly verified for the role in Se(Ⅳ) reduction and Cr(Ⅵ)resistance. Importantly, it is a promising strategy that B. wiedmannii ES2-45 is served as an efficient bacterium for SeNP synthesis, selenium biofortification and heavy metal bioremediation.展开更多
This letter critically comments on the article by Zheng et al investigating the role of aucubin in alleviating diabetic neuropathic pain(DNP).DNP arises from hyperglycaemia-induced nerve injury and microglial reprogra...This letter critically comments on the article by Zheng et al investigating the role of aucubin in alleviating diabetic neuropathic pain(DNP).DNP arises from hyperglycaemia-induced nerve injury and microglial reprogramming toward aerobic glycolysis.Aldose reductase(also known as AKR1B1)redirects excess glucose flux through the polyol pathway,thus increasing oxidative stress and inflammation.Zheng et al show that aucubin,a plant iridoid glycoside,reverses streptozotocin-induced mechanical and thermal hypersensitivity and anxiety-like behaviour in mice.Mechanistically,aucubin restores microglial morphology,reduces glycolytic flux,enhances oxidative phosphorylation and lowers tumour necrosis factor-α,interleukin(IL)-1βand IL-6 levels in spinal tissue and cultures of the BV-2 microglial cell line.Network pharmacology and molecular docking analyses identify AKR1B1 as a key target,confirmed by the fact that short hairpin RNA knockdown of AKR1B1 eliminates the effects of aucubin.Contrary to the other studies,this study uniquely implicates the polyol pathway in microglial immunometabolism.展开更多
Sennoside A(SA),a typical prodrug,exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases.Hydrolases have been identified,but reductases remain ...Sennoside A(SA),a typical prodrug,exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases.Hydrolases have been identified,but reductases remain unknown.By linking a photoreactive group to the SA scaffold,we synthesized a photoaffinity probe to covalently label SA reductases and identified SA reductases using activity-based protein profiling(ABPP).From lysates of an active strain,Bifidobacterium pseudocatenulatum(B.pseudocatenulatum),397 proteins were enriched and subsequently identified using mass spectrometry(MS).Among these proteins,chromate reductase/nicotinamide adenine dinucleotide(NADH)phosphate(NADPH)-dependent flavin mononucleotide(FMN)reductase/oxygen-insensitive NADPH nitroreductase(nfrA)was identified as a potent SA reductase through further bioinformatic analysis and The Universal Protein Resource(UniProt)database screening.We also determined that recombinant nfrA could reduce SA.Our study contributes to further illuminating mechanisms of SA transformation to rheinanthrone and simultaneously offers an effective method to identify gut bacterial reductases.展开更多
BACKGROUND Treatment of diabetic neuropathy is often limited by side effects.Aucubin,an iridoid glycoside derived from natural plants,exhibits notable anti-inflammatory and antioxidant properties.AIM To investigate th...BACKGROUND Treatment of diabetic neuropathy is often limited by side effects.Aucubin,an iridoid glycoside derived from natural plants,exhibits notable anti-inflammatory and antioxidant properties.AIM To investigate the effects of aucubin on diabetic neuropathic pain(DNP)and glycolysis and inflammation in microglia.METHODS Streptozotocin(STZ)was used to establish a DNP animal model.Blood glucose levels and body weight of mice were measured following STZ administration.Paw withdrawal threshold was calculated for mechanical allodynia.Paw withdrawal latency was recorded for thermal hyperalgesia.The open field test and elevated plus maze was used to assess locomotor activity and anxiety-like behavior.Western blotting was utilized for analysis of protein expression.Immunofluorescence staining was measured for morphometric analysis of microglia.Glycolysis and ATP synthesis in BV-2 cell lines were detected by metabolic extracellular flux analysis.The SwissTargetPrediction and STRING databases were used for comprehensive screening to identify potential target proteins for aucubin.The molecular docking between the possible target proteins and aucubin was investigated using Auto Dock Tool.The BV-2 cell line was transfected with lentiviral AKR1B1-shRNA to further ascertain the function of AKR1B1 in the impact of aucubin on aerobic glycolysis and inflammation during high glucose stimulation.RESULTS Aucubin significantly improved pain and anxiety-like behavior in STZ-induced diabetic mice and restored microglial aerobic glycolysis and inflammation.Several public databases and molecular docking studies suggested that AKR1B1,MMP2 and MMP9 are involved in the effect of aucubin on DNP.Aucubin failed to restore aerobic glycolysis and inflammation in the context of AKR1B1 deficiency.CONCLUSION Aucubin has potential as a therapeutic agent for alleviating DNP by inhibiting expression of AKR1B1.展开更多
Ulva prolifera,the primary causative species of green tide,has garnered significant attention due to its robust growth and reproductive capacity under high salt stress.However,there has been relatively little research...Ulva prolifera,the primary causative species of green tide,has garnered significant attention due to its robust growth and reproductive capacity under high salt stress.However,there has been relatively little research on the regulation of high salt stress in this species.In this study,we observed that high salt stress suppressed the growth of U.prolifera and leading to the nitric oxide(NO)accumulation,along with increased gene expression levels and enzyme activity of S-nitrosoglutathione reductase(GSNOR).Treatment with GSNOR inhibitor resulted in elevated NO levels under high salt stress,accompanied by reduced activity of antioxidant enzymes and decreased glutathione(GSH)accumulation,making U.prolifera more sensitive to high salt stress.Conversely,NO scavenger treatment not only reduced NO levels,but also weakened the high salt stress tolerance of U.prolifera.Furthermore,using tandem mass tags(TMT)switch analysis and mass spectrometry,we observed a significant increase in S nitrosylated protein levels in U.prolifera under high salt stress,with further augmentation upon GSNOR inhibitor treatment.We also found high salt stress induced S-nitrosylation(SNO)of glutathione reductase(GR),which is negatively regulated by GSNOR,resulting in increased GR activity.Our results show that under short-term high salt stress,the elevated expression level of GSNOR avoided excessive accumulation of NO,and a certain amount of NO enhanced the activity of antioxidant enzymes through SNO modification,which improve the high salt stress tolerance of U.prolifera,whereas under long-term high salt stress,excessive NO was toxic to U.prolifera.展开更多
Selenium(Se),an essential micronutrient among the 15 vital elements required for human physiology,exerts its biological functions primarily through its incorporation into selenoproteins.To date,approximately 25 seleno...Selenium(Se),an essential micronutrient among the 15 vital elements required for human physiology,exerts its biological functions primarily through its incorporation into selenoproteins.To date,approximately 25 selenoproteins have been characterized in mammalian systems,including glutathione peroxidase(GPX),thioredoxin reductase(TrxR),and iodothyronine deiodinases(DIOs),all of which exhibit indispensable physiological functions.展开更多
Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals,but their asymmetric synthesis remains challenging especially for those with multiple chiral centers.In ...Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals,but their asymmetric synthesis remains challenging especially for those with multiple chiral centers.In this study,IR104 from Streptomyces aureocirculatus was identified from 157 wild-type imine reductases for the synthesis of(S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide(antiepileptic drug Brivaracetam)via dynamic kinetic resolution reductive amination from ethyl 3-formylhexanoate and(S)-2-aminobutylamide with high diastereoselectivity.To further improve the catalytic efficiency of IR104,its mutant D191E/L195I/E253S/M258A(M3)was identified by saturation mutagenesis and iterative combinatorial mutagenesis,which exhibited a 102-fold increase in the catalytic efficiency relative to that of wild-type enzyme and high diastereoselectivity(98:2 d.r.).Crystal structural analysis and molecular dynamics simulations provided some insights into the molecular basis for the improved activity of the mutant enzyme.The imine reductase identified in this study could accept chiral amino amides/esters as amino donors for the dynamic kinetic resolution reductive amination of racemicα-substituted aldehydo-esters,expanding the substrate scope of imine reductases in the dynamic kinetic resolution-reductive amination.Finally,IR104-M3 was successfully used for the preparation of Brivaracetam at gram scale.Using this mutant,various N-substituted amino amides/esters with two chiral centers were also synthesized with up to 99:1 d.r.and 96%yields and subsequently converted intoγ-andδ-lactams,providing an efficient protocol for the synthesis of these important compounds via enzymatic dynamic kinetic resolution-reductive amination from simple building blocks.展开更多
Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researc...Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches.CYP80B,a family of cytochrome P450(CYP450)enzymes,plays a crucial role in BIA biosynthesis.Previously reported CYP80Bs are known to catalyze the 3′-hydroxylation of(S)-Nmethylcoclaurine,with the N-methyl group essential for catalytic activity.In this study,we successfully cloned a full-length CYP80B gene(St CYP80B)from Stephania tetrandra(S.tetrandra)and identified its function using a yeast heterologous expression system.Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that St CYP80B could catalyze Nmethylcoclaurine and coclaurine into their respective 3'-hydroxylated products.Notably,St CYP80B exhibited an expanded substrate selectivity compared to previously reported wildtype CYP80Bs,as it did not require an N-methyl group for hydroxylase activity.Furthermore,St CYP80B displayed a clear preference for the(S)-configuration.Co-expression of St CYP80B with the CYP450 reductases(CPRs,StCPR1,and StCPR2),also cloned from S.tetrandra,significantly enhanced the catalytic activity towards(S)-coclaurine.Site-directed mutagenesis of St CYP80B revealed that the residue H205 is crucial for coclaurine catalysis.Additionally,St CYP80B exhibited tissue-specific expression in plants.This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.展开更多
Background Saline-alkaline water aquaculture has become a key way to mitigate the reduction of freshwater aquaculture space and meet the increasing global demand for aquatic products.To enhance the comprehensive utili...Background Saline-alkaline water aquaculture has become a key way to mitigate the reduction of freshwater aquaculture space and meet the increasing global demand for aquatic products.To enhance the comprehensive utilization capability of saline-alkaline water,it is necessary to understand the regulatory mechanisms of aquatic animals coping with saline-alkaline water.In this study,our objective was to elucidate the function of proline metabolism in the alkaline adaptation of Nile tilapia(Oreochromis niloticus).Results Expose Nile tilapia to alkaline water of different alkalinity for 2 weeks to observe changes in its growth performance and proline metabolism.Meanwhile,to further clarify the role of proline metabolism,RNA interference experiments were conducted to disrupt the normal operation of proline metabolic axis by knocking down pycr(pyrroline-5-carboxylate reductases),the final rate-limiting enzyme in proline synthesis.The results showed that both the synthesis and degradation of proline were enhanced under carbonate alkalinity stress,and the environmental alkalinity impaired the growth performance of tilapia,and the higher the alkalinity,the greater the impairment.Moreover,environmental alkalinity caused oxidative stress in tilapia,enhanced ion transport,ammonia metabolism,and altered the intensity and form of energy metabolism in tilapia.When the expression level of the pycr gene decreased,the proline metabolism could not operate normally,and the ion transport,antioxidant defense system,and energy metabolism were severely damaged,ultimately leading to liver damage and a decreased survival rate of tilapia under alkalinity stress.Conclusions The results indicated that proline metabolism plays an important role in the alkaline adaptation of Nile tilapia and is a key regulatory process in various biochemical and physiological processes.展开更多
BACKGROUND Type 2 diabetes mellitus(T2DM)is associated with significant metabolic and renal complications,including diabetic nephropathy(DN).AIM To investigate the role of ribonucleotide reductase regulatory subunit M...BACKGROUND Type 2 diabetes mellitus(T2DM)is associated with significant metabolic and renal complications,including diabetic nephropathy(DN).AIM To investigate the role of ribonucleotide reductase regulatory subunit M2(RRM2)in T2DM and its potential involvement in renal injury through oxidative stress,apoptosis,and ferroptosis.METHODS A cross-sectional study was conducted,comprising 194 patients with T2DM and 120 healthy controls at our hospital between January 2022 and December 2023.The data were analyzed to ascertain the correlation between RRM2 levels and DN onset in patients with T2DM.The apoptosis rate,reactive oxygen species(ROS)levels,oxidative stress,cystine uptake,and ferrous ion(Fe2+)levels were quantified using the HK-2 cell lysates.Reverse transcription quantitative PCR and western blotting were used to assess mRNA and protein expression,respectively.RESULTS Serum RRM2 levels were significantly higher in T2DM patients than in controls(P<0.05)but declined in the macroalbuminuria subgroup.Receiver operating characteristic analysis identified 30 pg/mL as the optimal cut-off(area under the curve=0.958;sensitivity=86%;specificity=95%).RRM2 was negatively correlated with age,diabetes duration,systolic blood pressure,fasting blood glucose,glycosylated hemoglobin,serum creatinine,neutrophil gelatinase-associated lipocalin,kidney injury molecule-1,and malondialdehyde,and positively correlated with estimated glomerular filtration rate,glutathione(GSH),solute carrier family 7 member 11(SLC7A11),and GSH peroxidase 4(GPX4).Logistic regression confirmed RRM2 as an independent protective factor against DN[odds ratio(OR)=0.820,95%confidence interval(95%CI)=0.712-0.945,P=0.006].In vitro,RRM2 overexpression enhanced HK-2 cell proliferation,activated PI3K/Akt signaling,and reduced apoptosis,ROS,oxidative stress,and ferroptosis,accompanied by the restoration of GSH,Nrf2,SLC7A11,and GPX4.These protective effects were abolished by PI3K/Akt inhibition,highlighting RRM2’s renoprotective,pathway-dependent role.CONCLUSION These findings suggest that RRM2 plays a crucial protective role against diabetic renal injury by mitigating oxidative stress,apoptosis,and ferroptosis via PI3K/Akt activation.Serum RRM2 may serve as a novel biomarker for early DN detection,and therapeutic strategies targeting RRM2 may offer potential benefits in preventing diabetic kidney disease progression.展开更多
BACKGROUND Ferroptosis is an iron-dependent programmed non-apoptotic cell death characterized by the accumulation of free iron ions and lipid peroxidation.It is associated with the inactivation of glutathione peroxida...BACKGROUND Ferroptosis is an iron-dependent programmed non-apoptotic cell death characterized by the accumulation of free iron ions and lipid peroxidation.It is associated with the inactivation of glutathione peroxidase(GPX)and the accumulation of lipid peroxides within cells.Ferroptosis is closely related to the occurrence and development of hepatocellular carcinoma(HCC).Chlorogenic acid(CGA),an important bioactive component found in 61 traditional Chinese medicines such as Eucommia ulmoides,has been extensively studied for its effects on various malignant tumors.However,the specific role and potential mechanism of CGA in HCC remain unclear.AIM To elucidate the anti-tumor characteristics and potential mechanisms of CGA in inducing ferroptosis in HCC cells.METHODS The effects of CGA on the proliferation,migration,and invasion of HCC cells were evaluated through in vitro experiments.Bioinformatics analysis combined with network pharmacology was used to study the potential targets and molecular mechanisms of CGA intervention in HCC ferroptosis.In vitro experiments were conducted to verify and explore the anti-HCC effects and mechanisms of CGA through the ferroptosis pathway.RESULTS In vitro experiments showed that CGA dose-dependently inhibited the proliferation,invasion,and migration of HCC cells.Bioinformatics analysis combined with network pharmacology revealed that the pathway of CGA intervention in HCC cell ferroptosis was mainly enriched in the prostaglandin endoperoxide synthase 2(PTGS2)/aldoketo reductase family 1 member C3(AKR1C3)/GPX4 signaling pathway,which was associated with arachidonic acid.In vitro experiments further confirmed that CGA-induced ferroptosis in HCC cells was related to mitochondrial damage through the reprogramming of arachidonic acid metabolism by regulating the PTGS2/AKR1C3/GPX4 signaling pathway.CONCLUSION This study demonstrates that CGA inhibits HCC cell proliferation,migration,and invasion by inducing ferroptosis through the PTGS2/AKR1C3/GPX4 axis,suggesting its potential as a novel ferroptosis inducer or anti-HCC drug.展开更多
Methionine oxidation to methionine sulfoxide （MetSO） is reversed by two types of methionine sulfoxide reducrases （MSRs）, A and B, specific to the S- and R-diastereomers of MetSO, respectively. MSR genes are found ...Methionine oxidation to methionine sulfoxide （MetSO） is reversed by two types of methionine sulfoxide reducrases （MSRs）, A and B, specific to the S- and R-diastereomers of MetSO, respectively. MSR genes are found in most organisms from bacteria to human. In the current review, we first compare the organization of the MSR gene families in photosynthetic organisms from cyanobacteria to higher plants. The analysis reveals that MSRs constitute complex families in higher plants, bryophytes, and algae compared to cyanobacteria and all non-photosynthetic organisms. We also perform a classification, based on gene number and structure, position of redox-active cysteines and predicted sub-cellular localization. The various catalytic mechanisms and potential physiological electron donors involved in the regeneration of MSR activity are then de- scribed. Data available from higher plants reveal that MSRs fulfill an essential physiological function during environmental constraints through a role in protein repair and in protection against oxidative damage. Taking into consideration the ex- pression patterns of MSR genes in plants and the known roles of these genes in non-photosynthetic cells, other functions of MSRs are discussed during specific developmental stages and ageing in photosynthetic organisms.展开更多
文摘N-substituted furfurylamines(FAs)are valuable precursors for producing pharmacologically active compounds and polymers.However,enzymatic synthesis of the type of chemicals is still in its infancy.Here we report an imine reductase from Streptomyces albidoflavus(SaIRED)for the reductive amination of biobased furans.A simple,fast and interference-resistant high-throughput screening(HTS)method was developed,based on the coloration reaction of carbonyl compounds with 2,4-dinitrophenylhydrazine.The reductive amination activity of IREDs can be directly indicated by a colorimetric assay.With the reductive amination of furfural with allylamine as the model reaction,SaIRED with the activity of 4.8 U mg^(-1) was subjected to three rounds of protein engineering and screening by this HTS method,affording a high-activity tri-variant I127V/D241A/A242T(named M3,20.2 U mg^(-1)).The variant M3 showed broad substrate scope,and enabled efficient reductive amination of biobased furans with a variety of amines including small aliphatic amines and sterically hindered amines,giving the target FAs in yields up to>99%.In addition,other variants were identified for preparative-scale synthesis of commercially interesting amines such as N-2-(methylsulfonyl)ethyl-FA by the screen method,with isolated yields up to 87%and turnover numbers up to 9700 for enzyme.Gram-scale synthesis of N-allyl-FA,a valuable building block and potential polymer monomer,was implemented at 0.25 mol L^(-1) substrate loading by a whole-cell catalyst incorporating variant M3,with 4.7 g L^(-1) h^(-1) space-time yield and 91%isolated yield.
基金SN and GBR are supported by grants from the Science and Engineering Research Board,Government of India(EMR/2016/001984)Indian Council of Medical Research.
文摘Aldo-keto reductases(AKRs)are a superfamily of enzymes that play crucial roles in various cellular processes,including the metabolism of xenobiotics,steroids,and carbohydrates.A growing body of evidence has unveiled the involvement of AKRs in the development and progression of various cancers.AKRs are aberrantly expressed in a wide range of malignant tumors.Dysregulated expression of AKRs enables the acquisition of hallmark traits of cancer by activating oncogenic signaling pathways and contributing to chemoresistance.AKRs have emerged as promising oncotherapeutic targets given their pivotal role in cancer development and progression.Inhibition of aldose reductase(AR),either alone or in combination with chemotherapeutic drugs,has evolved as a pragmatic therapeutic option for cancer.Several classes of synthetic aldo-keto reductase(AKR)inhibitors have been developed as potential anticancer agents,some of which have shown promise in clinical trials.Many AKR inhibitors from natural sources also exhibit anticancer effects.Small molecule inhibitors targeting specific AKR isoforms have shown promise in preclinical studies.These inhibitors disrupt the activation of oncogenic signaling by modulating transcription factors and kinases and sensitizing cancer cells to chemotherapy.In this review,we discuss the physiological functions of human AKRs,the aberrant expression of AKRs in malignancies,the involvement of AKRs in the acquisition of cancer hallmarks,and the role of AKRs in oncogenic signaling,and drug resistance.Finally,the potential of aldose reductase inhibitors(ARIs)as anticancer drugs is summarized.
基金financially sponsored by the National Key Research and Development Program of China (2016YFA0204300, 2019YFA09005000)the National Natural Science Foundation of China (21536004, 21776085, 21871085)+2 种基金the Natural Science Foundation of Shanghai (18ZR1409900)Key Project of the Shanghai Science and Technology Committee (18DZ1112703)the Fundamental Research Funds for the Central Universities (WF1714026)。
文摘Optically pure(R)-γ-and(R)-δ-lactones can be prepared by intramolecular cyclization of chiral hydroxy acids/esters reduced asymmetrically from γ-and δ-keto acids/esters using Saccharomyces cerevisiae(S.cerevisiae) as a whole-cell biocatalyst.However,some of the enzymes catalyzing these reactions in S.cerevisiae are still unknown up to date.In this report,two carbonyl reductases,OdCRl and OdCR2,were successfully discovered,and cloned from S.cerevisiae using a genome-mining approach,and overexpressed in Escherichia coli(E.coli).Compared with OdCR1,OdCR2 can reduce 4-oxodecanoic acid and 5-oxodecanoic acid asymmetrically with higher stereoselectivity,generating(R)-γ-decalactone(99% ee) and(R)-δ-decalactone(98% ee) in 85% and 92%yields,respectively.This is the first report of native enzymes from S.cerevisiae for the enzymatic synthesis of chiral γ-and δ-lactones which is of wide uses in food and cosmetic industries.
文摘Introduction One of the major root alkaloids of the Indian medicinal plant Rauvolfia serpenlina Benth. ex Kurz is named ajmaline. The enzymatic biosynthesis of this alkaloid has been studied for a long time by our group As a result, a biosynthetic pathway has been established, in which about 10 enzymes participate, several of them belonging to the group of NADPH-dependent reductases. Started with the biosynthetic precursors tryptamine and secologanin,
基金Supported by the National Natural Science Foundation of China (31070042,30870039,30921065)partially by Open Funding Project of the National Key Laboratory of Biochemical Engineering,China (2010KF-2)
文摘The thermostability of three sulfur oxygenase reductases （SORs） was investigated from thermoacidophilic achaea Acidianus tengchongensis （SORAT） and Sulfolobus tokodaii （SORsT） as well as the moderately thermophilic bacterium Acidithiobacillus sp. SM-1 （SORsB）. The optimal temperatures for catalyzing sulfur oxidation were 80 ℃ （SORAT）, 85 ℃ （SORsT）, and 70 ℃ （SORsB）, respectively. The half-lives of the three SORs at their optimal catalytic conditions were 100 min （SORAT）, 58 min （SORsT）, and 37 min （SORsB）. In order to reveal the structural basis of the thermostability of these SORs, three-dimensional structural models of them were generated by homology modeling using the previously reported high-resolution X-ray structure of SORAA （from Acidianus ambivalens） as a template. The results suggest that thermostability was dependent on： （a） high number of the charged amino acid glutamic acid and the flexible amino acid proline, （b） low number of the therraolabile amino acid glutamine, （c） increased number of ion pairs, （d） decreased ratio of hydrophobie accessible solvent surface area （ASA） to charged ASA, and （e） increased volumes of the cavity. The number of cavities and the number of hydrogen bonds did not signifieantly affect the thermostability of SORs, whereas the cavity volumes increased as the thermal stability increased.
基金supported by the National Natural Science Foundation of China(No.41977029).
文摘Mining and tailings deposition can cause serious heavy metal(loids)pollution to the surrounding soil environment.Soil microorganisms adapt their metabolism to such conditions,driving alterations in soil function.This study aims to elucidate the response patterns of nitrogen-cycling microorganisms under long-term heavy metal(loids)exposure.The results showed that the diversity and abundance of nitrogen-cyclingmicroorganisms showed negative feedback to heavy metal(loids)concentrations.Denitrifying microorganisms were shown to be the dominant microorganisms with over 60%of relative abundance and a complex community structure including 27 phyla.Further,the key bacterial species in the denitrification process were calculated using a random forest model,where the top three key species(Pseudomonas stutzei,Sphingobium japonicum and Leifsonia rubra)were found to play a prominent role in nitrite reduction.Functional gene analysis and qPCR revealed that nirK,which is involved in nitrite reduction,significantly accumulated in the most metal-rich soil with the increase of absolute abundance of 63.86%.The experimental results confirmed that the activity of nitrite reductase(Nir)encoded by nirK in the soil was increased at high concentrations of heavy metal(loids).Partial least squares-path model identified three potential modes of nitrite reduction processes being stimulated by heavy metal(loids),the most prominent of which contributed to enhanced nirK abundance and soil Nir activity through positive stimulation of key species.The results provide new insights and preliminary evidence on the stimulation of nitrite reduction processes by heavy metal(loids).
基金Supported by Hebei Natural Science Foundation,No.H2022206539Hebei Provincial Government Funded Clinical Talents Training Project,No.ZF2023143.
文摘Surgical intervention is currently the primary treatment for hepatolithiasis;how-ever,some patients still experience residual stones and high recurrence rates after surgery.Cholesterol metabolism seems to play an important role in hepatoli-thiasis pathogenesis.A high cholesterol diet is one of the significant reasons for the increasing incidence of hepatolithiasis.Therefore,regular diet and appropriate medical intervention are crucial measures to prevent hepatolithiasis and reduce recurrence rate after surgery.Reducing dietary cholesterol and drugs that increase cholesterol stone solubility are key therapeutic approaches in treating hepato-lithiasis.This article discusses the cholesterol metabolic pathways related to the pathogenesis of hepatolithiasis,as well as food intake and targeted therapeutic drugs.
文摘Ethyl(R)-4-chloro-3-hydroxybutyrate((R)-CHBE),as a chiral intermediate,is widely used in the synthesis of various chiral drugs.In this study,we screened two aldo–keto reductases(LP-AKRs)from the probiotic Lactobacillus plantarum DSM20174,both with a molecular weight of approximately 31 kDa.Both enzymes could reduce 4-chloroacetoacetic acid ethyl ester(COBE)to produce(R)-CHBE with an enantioselectivity value of 99%.When determining the kinetic parameter,the K_(m),K_(cat),and V_(max)of LP-AKR5 and LP-AKR9 were 9.5 mM,7.6 U/mg,3.96 s^(-1)and 8.7 mM,8.59 U/mg,4.47 s^(-1),respectively.Both LP-AKR5 and LP-AKR9 had an optimal reaction pH of 6 and could maintain a high level of stability at pH 6,allowing them to perform well in an acidic environment.LP-AKR5 and LP-AKR9 had optimal reaction temperatures of 30℃and 40℃,respectively.Metal ions had minimal influence on LP-AKR5 and LP-AKR9 enzyme activities.This series of enzymatic properties showed that LP-AKR5 and LP-AKR9 mined from Lactobacillus plantarum DSM20174 could asym-metrically catalyze the synthesis of(R)-CHBE under weakly acidic circumstances,which could maintain product stability and provide a good foundation for industrial production.
基金supported by the National Natural Science Foundation of China(No.32370130).
文摘Microbial selenite(Se(Ⅳ)) reduction to elemental selenium nanoparticles(SeNPs) and chromate(Cr(Ⅵ)) remediation constitute effective detoxification processes. The strain Bacillus wiedmannii ES2-45 can efficiently reduce 5 mmol/L Se(Ⅳ) into SeNPs over 14 h and 1 mmol/L Cr(Ⅵ) within 36 h. Proteomic analysis and qRT-PCR revealed that reductases, including dithiol oxidoreductase(DsbA), Fe-S biosynthesis domain-containing protein(Fes),and aldose-6-phosphate reductase(Aldo), and bacillithiol(BSH) were involved in Se(Ⅳ) reduction. Heterologous expression further confirmed the Se(Ⅳ)-reducing function of three genes and enhanced the Se(Ⅳ) and Cr(Ⅵ) resistance ability. Moreover, the difference between Se(Ⅳ) treatments and controls both intra-and extra-cellular BSH concentration per biomass indicated that BSH contributes to Se(Ⅳ) reduction. Selenium-fortified rice was obtained by foliar spray of SeNPs synthesized by the strain ES2-45. To the best of our knowledge, DsbA and Aldo reductase were firstly verified for the role in Se(Ⅳ) reduction and Cr(Ⅵ)resistance. Importantly, it is a promising strategy that B. wiedmannii ES2-45 is served as an efficient bacterium for SeNP synthesis, selenium biofortification and heavy metal bioremediation.
基金Supported by the Top-level Talents Support Program of Yangzhou University“Lv Yang Jin Feng”Outstanding Doctor of Yangzhou,No.YZLYJFJH2023YXBS169Natural Science Foundation of Jiangsu Province,No.BK20240907.
文摘This letter critically comments on the article by Zheng et al investigating the role of aucubin in alleviating diabetic neuropathic pain(DNP).DNP arises from hyperglycaemia-induced nerve injury and microglial reprogramming toward aerobic glycolysis.Aldose reductase(also known as AKR1B1)redirects excess glucose flux through the polyol pathway,thus increasing oxidative stress and inflammation.Zheng et al show that aucubin,a plant iridoid glycoside,reverses streptozotocin-induced mechanical and thermal hypersensitivity and anxiety-like behaviour in mice.Mechanistically,aucubin restores microglial morphology,reduces glycolytic flux,enhances oxidative phosphorylation and lowers tumour necrosis factor-α,interleukin(IL)-1βand IL-6 levels in spinal tissue and cultures of the BV-2 microglial cell line.Network pharmacology and molecular docking analyses identify AKR1B1 as a key target,confirmed by the fact that short hairpin RNA knockdown of AKR1B1 eliminates the effects of aucubin.Contrary to the other studies,this study uniquely implicates the polyol pathway in microglial immunometabolism.
基金supported by the National Natural Science Foundation of China(Grant Nos.:U21A20407 and 81973467)Beijing Natural Science Foundation,China(Grant No.:7222276).
文摘Sennoside A(SA),a typical prodrug,exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases.Hydrolases have been identified,but reductases remain unknown.By linking a photoreactive group to the SA scaffold,we synthesized a photoaffinity probe to covalently label SA reductases and identified SA reductases using activity-based protein profiling(ABPP).From lysates of an active strain,Bifidobacterium pseudocatenulatum(B.pseudocatenulatum),397 proteins were enriched and subsequently identified using mass spectrometry(MS).Among these proteins,chromate reductase/nicotinamide adenine dinucleotide(NADH)phosphate(NADPH)-dependent flavin mononucleotide(FMN)reductase/oxygen-insensitive NADPH nitroreductase(nfrA)was identified as a potent SA reductase through further bioinformatic analysis and The Universal Protein Resource(UniProt)database screening.We also determined that recombinant nfrA could reduce SA.Our study contributes to further illuminating mechanisms of SA transformation to rheinanthrone and simultaneously offers an effective method to identify gut bacterial reductases.
基金Supported by National Natural Science Foundation of China,No.82001424.
文摘BACKGROUND Treatment of diabetic neuropathy is often limited by side effects.Aucubin,an iridoid glycoside derived from natural plants,exhibits notable anti-inflammatory and antioxidant properties.AIM To investigate the effects of aucubin on diabetic neuropathic pain(DNP)and glycolysis and inflammation in microglia.METHODS Streptozotocin(STZ)was used to establish a DNP animal model.Blood glucose levels and body weight of mice were measured following STZ administration.Paw withdrawal threshold was calculated for mechanical allodynia.Paw withdrawal latency was recorded for thermal hyperalgesia.The open field test and elevated plus maze was used to assess locomotor activity and anxiety-like behavior.Western blotting was utilized for analysis of protein expression.Immunofluorescence staining was measured for morphometric analysis of microglia.Glycolysis and ATP synthesis in BV-2 cell lines were detected by metabolic extracellular flux analysis.The SwissTargetPrediction and STRING databases were used for comprehensive screening to identify potential target proteins for aucubin.The molecular docking between the possible target proteins and aucubin was investigated using Auto Dock Tool.The BV-2 cell line was transfected with lentiviral AKR1B1-shRNA to further ascertain the function of AKR1B1 in the impact of aucubin on aerobic glycolysis and inflammation during high glucose stimulation.RESULTS Aucubin significantly improved pain and anxiety-like behavior in STZ-induced diabetic mice and restored microglial aerobic glycolysis and inflammation.Several public databases and molecular docking studies suggested that AKR1B1,MMP2 and MMP9 are involved in the effect of aucubin on DNP.Aucubin failed to restore aerobic glycolysis and inflammation in the context of AKR1B1 deficiency.CONCLUSION Aucubin has potential as a therapeutic agent for alleviating DNP by inhibiting expression of AKR1B1.
基金Supported by the National Natural Science Foundation of China(No.42276100)the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)。
文摘Ulva prolifera,the primary causative species of green tide,has garnered significant attention due to its robust growth and reproductive capacity under high salt stress.However,there has been relatively little research on the regulation of high salt stress in this species.In this study,we observed that high salt stress suppressed the growth of U.prolifera and leading to the nitric oxide(NO)accumulation,along with increased gene expression levels and enzyme activity of S-nitrosoglutathione reductase(GSNOR).Treatment with GSNOR inhibitor resulted in elevated NO levels under high salt stress,accompanied by reduced activity of antioxidant enzymes and decreased glutathione(GSH)accumulation,making U.prolifera more sensitive to high salt stress.Conversely,NO scavenger treatment not only reduced NO levels,but also weakened the high salt stress tolerance of U.prolifera.Furthermore,using tandem mass tags(TMT)switch analysis and mass spectrometry,we observed a significant increase in S nitrosylated protein levels in U.prolifera under high salt stress,with further augmentation upon GSNOR inhibitor treatment.We also found high salt stress induced S-nitrosylation(SNO)of glutathione reductase(GR),which is negatively regulated by GSNOR,resulting in increased GR activity.Our results show that under short-term high salt stress,the elevated expression level of GSNOR avoided excessive accumulation of NO,and a certain amount of NO enhanced the activity of antioxidant enzymes through SNO modification,which improve the high salt stress tolerance of U.prolifera,whereas under long-term high salt stress,excessive NO was toxic to U.prolifera.
基金Financial support from the Science and Technology Innovation Program of Hunan Province(No.2022RC4044)。
文摘Selenium(Se),an essential micronutrient among the 15 vital elements required for human physiology,exerts its biological functions primarily through its incorporation into selenoproteins.To date,approximately 25 selenoproteins have been characterized in mammalian systems,including glutathione peroxidase(GPX),thioredoxin reductase(TrxR),and iodothyronine deiodinases(DIOs),all of which exhibit indispensable physiological functions.
文摘Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals,but their asymmetric synthesis remains challenging especially for those with multiple chiral centers.In this study,IR104 from Streptomyces aureocirculatus was identified from 157 wild-type imine reductases for the synthesis of(S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide(antiepileptic drug Brivaracetam)via dynamic kinetic resolution reductive amination from ethyl 3-formylhexanoate and(S)-2-aminobutylamide with high diastereoselectivity.To further improve the catalytic efficiency of IR104,its mutant D191E/L195I/E253S/M258A(M3)was identified by saturation mutagenesis and iterative combinatorial mutagenesis,which exhibited a 102-fold increase in the catalytic efficiency relative to that of wild-type enzyme and high diastereoselectivity(98:2 d.r.).Crystal structural analysis and molecular dynamics simulations provided some insights into the molecular basis for the improved activity of the mutant enzyme.The imine reductase identified in this study could accept chiral amino amides/esters as amino donors for the dynamic kinetic resolution reductive amination of racemicα-substituted aldehydo-esters,expanding the substrate scope of imine reductases in the dynamic kinetic resolution-reductive amination.Finally,IR104-M3 was successfully used for the preparation of Brivaracetam at gram scale.Using this mutant,various N-substituted amino amides/esters with two chiral centers were also synthesized with up to 99:1 d.r.and 96%yields and subsequently converted intoγ-andδ-lactams,providing an efficient protocol for the synthesis of these important compounds via enzymatic dynamic kinetic resolution-reductive amination from simple building blocks.
文摘Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches.CYP80B,a family of cytochrome P450(CYP450)enzymes,plays a crucial role in BIA biosynthesis.Previously reported CYP80Bs are known to catalyze the 3′-hydroxylation of(S)-Nmethylcoclaurine,with the N-methyl group essential for catalytic activity.In this study,we successfully cloned a full-length CYP80B gene(St CYP80B)from Stephania tetrandra(S.tetrandra)and identified its function using a yeast heterologous expression system.Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that St CYP80B could catalyze Nmethylcoclaurine and coclaurine into their respective 3'-hydroxylated products.Notably,St CYP80B exhibited an expanded substrate selectivity compared to previously reported wildtype CYP80Bs,as it did not require an N-methyl group for hydroxylase activity.Furthermore,St CYP80B displayed a clear preference for the(S)-configuration.Co-expression of St CYP80B with the CYP450 reductases(CPRs,StCPR1,and StCPR2),also cloned from S.tetrandra,significantly enhanced the catalytic activity towards(S)-coclaurine.Site-directed mutagenesis of St CYP80B revealed that the residue H205 is crucial for coclaurine catalysis.Additionally,St CYP80B exhibited tissue-specific expression in plants.This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.
基金supported by grants from the National Natural Science Foundation of China(No.32172946)the Young Elite Scientists Sponsorship Program by CAST(2022QNRC001)+1 种基金the Shanghai Rising-Star Program(2022)the Fundamental Research Funds for the Central Universities,ECNU。
文摘Background Saline-alkaline water aquaculture has become a key way to mitigate the reduction of freshwater aquaculture space and meet the increasing global demand for aquatic products.To enhance the comprehensive utilization capability of saline-alkaline water,it is necessary to understand the regulatory mechanisms of aquatic animals coping with saline-alkaline water.In this study,our objective was to elucidate the function of proline metabolism in the alkaline adaptation of Nile tilapia(Oreochromis niloticus).Results Expose Nile tilapia to alkaline water of different alkalinity for 2 weeks to observe changes in its growth performance and proline metabolism.Meanwhile,to further clarify the role of proline metabolism,RNA interference experiments were conducted to disrupt the normal operation of proline metabolic axis by knocking down pycr(pyrroline-5-carboxylate reductases),the final rate-limiting enzyme in proline synthesis.The results showed that both the synthesis and degradation of proline were enhanced under carbonate alkalinity stress,and the environmental alkalinity impaired the growth performance of tilapia,and the higher the alkalinity,the greater the impairment.Moreover,environmental alkalinity caused oxidative stress in tilapia,enhanced ion transport,ammonia metabolism,and altered the intensity and form of energy metabolism in tilapia.When the expression level of the pycr gene decreased,the proline metabolism could not operate normally,and the ion transport,antioxidant defense system,and energy metabolism were severely damaged,ultimately leading to liver damage and a decreased survival rate of tilapia under alkalinity stress.Conclusions The results indicated that proline metabolism plays an important role in the alkaline adaptation of Nile tilapia and is a key regulatory process in various biochemical and physiological processes.
文摘BACKGROUND Type 2 diabetes mellitus(T2DM)is associated with significant metabolic and renal complications,including diabetic nephropathy(DN).AIM To investigate the role of ribonucleotide reductase regulatory subunit M2(RRM2)in T2DM and its potential involvement in renal injury through oxidative stress,apoptosis,and ferroptosis.METHODS A cross-sectional study was conducted,comprising 194 patients with T2DM and 120 healthy controls at our hospital between January 2022 and December 2023.The data were analyzed to ascertain the correlation between RRM2 levels and DN onset in patients with T2DM.The apoptosis rate,reactive oxygen species(ROS)levels,oxidative stress,cystine uptake,and ferrous ion(Fe2+)levels were quantified using the HK-2 cell lysates.Reverse transcription quantitative PCR and western blotting were used to assess mRNA and protein expression,respectively.RESULTS Serum RRM2 levels were significantly higher in T2DM patients than in controls(P<0.05)but declined in the macroalbuminuria subgroup.Receiver operating characteristic analysis identified 30 pg/mL as the optimal cut-off(area under the curve=0.958;sensitivity=86%;specificity=95%).RRM2 was negatively correlated with age,diabetes duration,systolic blood pressure,fasting blood glucose,glycosylated hemoglobin,serum creatinine,neutrophil gelatinase-associated lipocalin,kidney injury molecule-1,and malondialdehyde,and positively correlated with estimated glomerular filtration rate,glutathione(GSH),solute carrier family 7 member 11(SLC7A11),and GSH peroxidase 4(GPX4).Logistic regression confirmed RRM2 as an independent protective factor against DN[odds ratio(OR)=0.820,95%confidence interval(95%CI)=0.712-0.945,P=0.006].In vitro,RRM2 overexpression enhanced HK-2 cell proliferation,activated PI3K/Akt signaling,and reduced apoptosis,ROS,oxidative stress,and ferroptosis,accompanied by the restoration of GSH,Nrf2,SLC7A11,and GPX4.These protective effects were abolished by PI3K/Akt inhibition,highlighting RRM2’s renoprotective,pathway-dependent role.CONCLUSION These findings suggest that RRM2 plays a crucial protective role against diabetic renal injury by mitigating oxidative stress,apoptosis,and ferroptosis via PI3K/Akt activation.Serum RRM2 may serve as a novel biomarker for early DN detection,and therapeutic strategies targeting RRM2 may offer potential benefits in preventing diabetic kidney disease progression.
基金the National Natural Science Foundation of China,No.82074425Natural Foundation of Hunan Province,No.2023JJ30364 and No.2023JJ30361+1 种基金Hunan Provincial Key R&D Program,No.2023SK2057Key Project of Hunan Provincial Administration of Traditional Chinese Medicine,No.A2023042.
文摘BACKGROUND Ferroptosis is an iron-dependent programmed non-apoptotic cell death characterized by the accumulation of free iron ions and lipid peroxidation.It is associated with the inactivation of glutathione peroxidase(GPX)and the accumulation of lipid peroxides within cells.Ferroptosis is closely related to the occurrence and development of hepatocellular carcinoma(HCC).Chlorogenic acid(CGA),an important bioactive component found in 61 traditional Chinese medicines such as Eucommia ulmoides,has been extensively studied for its effects on various malignant tumors.However,the specific role and potential mechanism of CGA in HCC remain unclear.AIM To elucidate the anti-tumor characteristics and potential mechanisms of CGA in inducing ferroptosis in HCC cells.METHODS The effects of CGA on the proliferation,migration,and invasion of HCC cells were evaluated through in vitro experiments.Bioinformatics analysis combined with network pharmacology was used to study the potential targets and molecular mechanisms of CGA intervention in HCC ferroptosis.In vitro experiments were conducted to verify and explore the anti-HCC effects and mechanisms of CGA through the ferroptosis pathway.RESULTS In vitro experiments showed that CGA dose-dependently inhibited the proliferation,invasion,and migration of HCC cells.Bioinformatics analysis combined with network pharmacology revealed that the pathway of CGA intervention in HCC cell ferroptosis was mainly enriched in the prostaglandin endoperoxide synthase 2(PTGS2)/aldoketo reductase family 1 member C3(AKR1C3)/GPX4 signaling pathway,which was associated with arachidonic acid.In vitro experiments further confirmed that CGA-induced ferroptosis in HCC cells was related to mitochondrial damage through the reprogramming of arachidonic acid metabolism by regulating the PTGS2/AKR1C3/GPX4 signaling pathway.CONCLUSION This study demonstrates that CGA inhibits HCC cell proliferation,migration,and invasion by inducing ferroptosis through the PTGS2/AKR1C3/GPX4 axis,suggesting its potential as a novel ferroptosis inducer or anti-HCC drug.
文摘Methionine oxidation to methionine sulfoxide （MetSO） is reversed by two types of methionine sulfoxide reducrases （MSRs）, A and B, specific to the S- and R-diastereomers of MetSO, respectively. MSR genes are found in most organisms from bacteria to human. In the current review, we first compare the organization of the MSR gene families in photosynthetic organisms from cyanobacteria to higher plants. The analysis reveals that MSRs constitute complex families in higher plants, bryophytes, and algae compared to cyanobacteria and all non-photosynthetic organisms. We also perform a classification, based on gene number and structure, position of redox-active cysteines and predicted sub-cellular localization. The various catalytic mechanisms and potential physiological electron donors involved in the regeneration of MSR activity are then de- scribed. Data available from higher plants reveal that MSRs fulfill an essential physiological function during environmental constraints through a role in protein repair and in protection against oxidative damage. Taking into consideration the ex- pression patterns of MSR genes in plants and the known roles of these genes in non-photosynthetic cells, other functions of MSRs are discussed during specific developmental stages and ageing in photosynthetic organisms.
