2-Oxoglutarate(2OG)-dependent dioxygenases(2-ODDs)are omnipresent iron-containing non-heme enzymes that catalyze various oxidation-reduction reactions in plant growth and development,nucleic acid modification and seco...2-Oxoglutarate(2OG)-dependent dioxygenases(2-ODDs)are omnipresent iron-containing non-heme enzymes that catalyze various oxidation-reduction reactions in plant growth and development,nucleic acid modification and secondary metabolism.We systematically summarized recent research on the oxidative modifications of plant 2-ODDs and related enzymes,their vital importance in the biosynthesis of plant special metabolites,and their catalytic specificity/flexibility,and discussed the potential of 2-ODD as a new approach for the identification of pivotal genes and the elucidation of biosynthetic pathway.展开更多
Enantioselective cis-dihydroxylation of alkenes represents an ideal route to synthesize enantioenriched syn-2,3-dihydroxy esters that are important structural motifs in numerous biologically and pharmaceutically relev...Enantioselective cis-dihydroxylation of alkenes represents an ideal route to synthesize enantioenriched syn-2,3-dihydroxy esters that are important structural motifs in numerous biologically and pharmaceutically relevant molecules.Bioinspired nonheme iron-catalyzed enantioselective cis-dihydroxylation meets the requirement of the modern synthetic chemistry from the atomic economy,green chemistry,and sustainable development perspectives.However,nonheme iron-catalyzed enantioselective cis-dihydroxylation is much underdeveloped because of the formidable challenges of controlling chemo-and enantioselectivities and product selectivity caused by the competitive epoxidation,cis-dihydroxylation,and overoxidation reactions.Herein,we disclose the fabrication of a biologically inspired nonheme iron complex-catalyzed enantioselective cis-dihydroxylation of multisubstituted acrylates using hydrogen peroxide(H_(2)O_(2))as the terminal oxidant by controlling the non-ligating or weakly ligating counterions of iron(Ⅱ)complexes,demonstrating a dramatic counteranion effect on the enantioselective cisdihydroxylation of olefins by H_(2)O_(2) catalyzed by nonheme iron complexes.A range of structurally disparate alkenes were transformed to the corresponding syn-2,3-dihydroxy esters in practically useful yields with exquisite chemo-and enantioselectivities(up to 99% ee).Given the mild and benign nature of this biologically inspired oxidation system as well as the ubiquity and synthetic utility of enantioenriched syn-2,3-dihydroxy esters as pharmaceuticals candidates and natural products,we expect that this strategy could serve as a promising complement to the well-known Sharpless asymmetric dihydroxylation,which is the chemical reaction of an alkene with OsO_(4) to produce a vicinal diol.展开更多
The carotenoid-derived volatileβ-ionone makes an important contribution to tea fragrance.Here,we qualitatively and quantitatively analysed 15 carotenoids in tea leaves of 13 cultivars by UHPLC-APCI-MS/MS.The 13 culti...The carotenoid-derived volatileβ-ionone makes an important contribution to tea fragrance.Here,we qualitatively and quantitatively analysed 15 carotenoids in tea leaves of 13 cultivars by UHPLC-APCI-MS/MS.The 13 cultivars were divided into two groups by PCA(Principal Component Analysis)clustering analysis of their carotenoid content,and OPLS-DA(Orthogonal projections to latent structures)indicated that the levels ofβ-carotene(VIP=2.89)and lutein(VIP=2.30)were responsible for much of the variation between the two groups.Interestingly,theβ-carotene toβ-ionone conversion rates in Group 1 were higher than in Group 2,while theβ-carotene content was significantly lower in Group 1 than in Group 2.Theβ-ionone content was significantly higher in Group 1.Pearson Correlation Coefficient calculation between the transcription level of candidate genes(CsCCD1 and CsCCD4)and the accumulation ofβ-ionone indicated that CsCCD1 may involve in the formation ofβ-ionone in 13 cultivars.Prokaryotic expression and in vitro enzyme activity assays showed that‘Chuanhuang 1’had an amino acid mutation in carotenoid cleavage dioxygenases 1(CsCCD1)compared with‘Shuchazao’,resulting in a significantly higherβ-ionone content in‘Chuanhuang 1’.Sequence analysis showed that‘Chuanhuang 1’and‘Huangdan’had different CsCCD1 promoter sequences,leading to significantly higher CsCCD1 expression andβ-ionone accumulation in‘Chuanhuang 1’.These results indicated that the promoter and coding sequence diversity of CsCCD1 might contribute to the differential accumulation ofβ-ionone in different tea cultivars.展开更多
Abscisic acid(ABA)is a plant hormone that plays an important role in plant development and abiotic stress.The 9-cis-epoxycarotenoid dioxygenase(NCED)is a key rate-limiting enzyme in the ABA biosynthetic pathway.The ph...Abscisic acid(ABA)is a plant hormone that plays an important role in plant development and abiotic stress.The 9-cis-epoxycarotenoid dioxygenase(NCED)is a key rate-limiting enzyme in the ABA biosynthetic pathway.The physiological and molecular mechanisms of NCED regulating plant development and abiotic stress tolerance have been reported in many plant species,but gene function of RiNCEDs in Rubus idaeus L.is rarely reported.In this study,the open reading frame(ORF)sequence of RiNCED2 in red raspberry fruit was isolated and the function of this gene under abiotic stress was investigated.While RiNCED2 was induced by cold,high salinity,drought and ABA,it was highly expressed in new leaves as measured by real-time qPCR.Overexpression of RiNCED2 in Arabidopsis under both high-salt and cold stress increased ABA content,demonstrating that RiNCED2 was involved in ABA biosynthesis.Meanwhile,the leaf wilting degree of transgenic Arabidopsis was less,while the content of malondialdehyde(MDA)was significantly reduced,and the chlorophyll content,proline content,peroxidase(POD),catalase(CAT)and superoxide dismutase(SOD)activities were significantly increased.These results indicated that overexpression of RiNCED2 enhanced the resistance of transgenic Arabidopsis to high salt and cold.展开更多
Carotenoid cleavage dioxygenases(CCDs) are a class of enzymes in plants involved in the biosynthesis of apocarotenoids,such as phytohormones,flavour compounds,and other compounds with yet unknown functions.To date,sev...Carotenoid cleavage dioxygenases(CCDs) are a class of enzymes in plants involved in the biosynthesis of apocarotenoids,such as phytohormones,flavour compounds,and other compounds with yet unknown functions.To date,several CCDs have been functionally characterised in plants,but little is known about the CCD4 members.A carotenoid cleavage dioxygenase 4 gene(LcCCD4) was isolated from the leaves of wolfberry(Lycium chinense) to gain insight into its biological function.Multiple sequence alignment and phylogenetic analyses showed that the deduced amino acid sequence of LcCCD4 shares high homology with that of CCD4 proteins from other plants.Expression analysis using semi-quantitative polymerase chain reaction revealed that LcCCD4 was strongly expressed in leaves and flowers and that the expression level was in accordance with β-carotene concentration.LcCCD4 transcripts in fruits tended to decrease as carotenoids accumulated.Recombinant expression of LcCCD4 cleaved β-carotene to produce P-ionone in in vivo assays.These results show that LcCCD4 is a CCD gene that may be involved in producing aromatic apocarotenoids in leaves and flowers,whereas it may be involved in controlling carotenoid accumulation in fruits.展开更多
A novel biphenyl-degrading bacterial strain LA-4 was isolated from activated sludge. It was identified as Dyella ginsengisoli according to phylogenetic similarity of 16S rRNA gene sequence. This isolate could utilize ...A novel biphenyl-degrading bacterial strain LA-4 was isolated from activated sludge. It was identified as Dyella ginsengisoli according to phylogenetic similarity of 16S rRNA gene sequence. This isolate could utilize biphenyl as sole source of carbon and energy, which degraded over 95 mg/L biphenyl within 36 h. The major metabolites formed from biphenyl, such as 2-hydroxy-6-oxo-6- phenylhexa-2,4-dienoic acid (HOPDA) and benzoic acid, were identified by LC-MS. The crude cell extract of strain LA-4 exhibited the activity of 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD) and the kinetic parameters Km was 26.48 μmol/L and Vmax was 8.12 U/mg protein. A conserved region of the biphenyl dioxygenase gene bphA1 of strain LA-4 was amplified by PCR and confirmed by DNA sequencing.展开更多
Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors of the gastrointestinal tract.At the molecular level,GISTs can be categorized into two groups based on the causative oncogenic mutations.App...Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors of the gastrointestinal tract.At the molecular level,GISTs can be categorized into two groups based on the causative oncogenic mutations.Approximately 85%of GISTs are caused by gain-of-function mutations in the tyrosine kinase receptor KIT or platelet-derived growth factor receptor alpha(PDGFRA).The remaining GISTs,referred to as wild-type(WT)GISTs,are often deficient in succinate dehydrogenase complex(SDH),a key metabolic enzyme complex in the tricarboxylic acid(TCA)cycle and electron transport chain.SDH deficiency leads to the accumulation of succinate,a metabolite produced by the TCA cycle.Succinate inhibitsα-ketoglutarate-dependent dioxygenase family enzymes,which comprise approximately 60 members and regulate key aspects of tumorigenesis such as DNA and histone demethylation,hypoxia responses,and m6A mRNA modification.For this reason,succinate and metabolites with similar structures,such as D-2-hydroxyglutarate and fumarate,are considered oncometabolites.In this article,we review recent advances in the understanding of how metabolic enzyme mutations and oncometabolites drive human cancer with an emphasis on SDH mutations and succinate in WT GISTs.展开更多
High-tillering dwarf mutant gsor23 was generated from an indica rice variety Indica9 radiatied by y-ray. Genetic analysis showed that this phenotype was controlled by one single recessive gene, which was mapped within...High-tillering dwarf mutant gsor23 was generated from an indica rice variety Indica9 radiatied by y-ray. Genetic analysis showed that this phenotype was controlled by one single recessive gene, which was mapped within a physical distance of 386 kb between two insertion-deletion (InDel) markers CI-WT2 and C1-WT4 on the long arm of chromosome 1. There is a known gene DIO within this region, the mutation of which causes high-tillering in rice. Sequence analysis of the DIO allele in gsor23 revealed that the base cytosine (C) at the 404th position in the coding region was deleted, which would cause frameshift mutation after the 134th amino acids. The mutation site and indica background of gsor23 were different from the previously reported japonica mutants d10-1 and d10-2. Therefore, gsor23 is a novel allelic mutant of D10 which encodes the carotenoid-cleaving dioxygenase 8 (CCD8), a key enzyme involved in the biosynthesis of the new plant hormone strigolactones (SLs). After treatment with GR24, a synthetic analogue of SLs, the high-tillering phenotype of gsor23 was restored to normal. Real-time RT-PCR analysis showed that D10 expression was high in roots, but low in leaves. Compared with the wild type Indica9, the expression of the SL biosynthesis gene DIO was upregulated, while genes likely involved in the SL signal transduction pathway such as D3 and D14 were down-regulated in the gsor23 mutant.展开更多
Lutein,a type of carotenoids,is found to delay the onset and progression of age-related macular degeneration(AMD).Several lutein supplementation studies showed that after an initial increase,lutein serum levels demons...Lutein,a type of carotenoids,is found to delay the onset and progression of age-related macular degeneration(AMD).Several lutein supplementation studies showed that after an initial increase,lutein serum levels demonstrated a subsequent decrease despite continuous supplementation.In this systematic literature review,this obscure phenomenon was tried to be explained.The subsequent drop in lutein levels was postulated due to down-regulation of lutein receptors scavenger receptor class B typeⅠ(SR-BI)in the gastrointestinal tract,upregulation of lutein degrading enzymeβ-carotene dioxygenase(BCDO2),or perhaps a combination of both.Some single nucleotides polymorphisms(SNPs)that could have influence on the occurrence of this phenomenon.To date,an exact scientific explanation for this phenomenon has not been established.Further research is needed to investigate this phenomenon in depth to reach an irrefutable explanation,giving that lutein is proven to be effective in delaying the onset and progression of AMD and its metabolism in the human body becomes of equal importance.展开更多
To find new extradiol dioxygenases(EDOs,EC 1.13.11.2),a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity...To find new extradiol dioxygenases(EDOs,EC 1.13.11.2),a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity.A novel EDO,designated as BphC_A,was identified and heterologously expressed in Escherichia coli.The deduced amino acid sequence of BphC_A exhibited a homology of less than 60% with other known EDOs.Phylogenetic analysis of BphC_A suggests that the protein is a novel member of the EDO family.The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol,the preferred substrate of other known EDOs.The optimum activity of purified BphC_A occurred at pH=8.5 and 35 °C,and BphC_A showed more than 40% of its initial activity at 5 °C.The activity of purified BphC_A was significantly induced by Mn^2+ and slightly reduced by Al^3+,Cu^2+ and Zn^2+.展开更多
The Pseudomonas putida TOL plasmid gene xy1E,whichproduces catechol 2,3-dioxygenase (CatO<sub>2</sub>ase;catechol:oxygen 2,3-oxidoreductase (decyclizing),EC 1.13.11.2 )was used as a target gene to stud...The Pseudomonas putida TOL plasmid gene xy1E,whichproduces catechol 2,3-dioxygenase (CatO<sub>2</sub>ase;catechol:oxygen 2,3-oxidoreductase (decyclizing),EC 1.13.11.2 )was used as a target gene to study the mechanisms ofmutation and induced mutation frequency in展开更多
To construct a high-level expression of xylE gene in E. coli for quick detection of environmental pollution of aromatic compounds. Methods and Results: XylE gene coding for the catechol 2, 3 dioxygenase (CatO2ase ) wa...To construct a high-level expression of xylE gene in E. coli for quick detection of environmental pollution of aromatic compounds. Methods and Results: XylE gene coding for the catechol 2, 3 dioxygenase (CatO2ase ) was amplified from the recombinant plasmid pTG402 by using PCR technique and was subcloned into pUC118N and pUC119N. The single stranded recombinant phage DNA from the transformed E. coli MV1184 cells was used for sequencing. The sequence of xylE gene was proved to be the same as reported. The gene was then subcloned into the high expression plasmid pJLA503, its expression amount being about 34. 2% of the total bacterial proteins. Conclusion: xylE gene is highly expressed in host E. coli TG1.展开更多
In plants, demethylation of 5-methylcytosine (5 mC) residues is controlled by DNA glycosylases, while in mammals it requires oxidation of 5 mC by TET proteins, a group of Fe(II)/2-oxoglutaratedependent dioxygenases. W...In plants, demethylation of 5-methylcytosine (5 mC) residues is controlled by DNA glycosylases, while in mammals it requires oxidation of 5 mC by TET proteins, a group of Fe(II)/2-oxoglutaratedependent dioxygenases. We analysed the effects of expressing the C-terminal catalytic domain of the human TET3 gene (TET3c) in Arabidopsis thaliana, using an rDNA region as a methylation reporter. In TET3c transformants, epialleles with hypomethylation or hypermethylation patterns can be induced, which is each stably retained in progeny lines even after removal of the TET3c transgene. In TET3c transformants, 5-hydroxymethylcytosine (5 hmC) marks are detected, indicative of the oxidative activity of the transgenic enzyme. 5-formylcytosine (5 fC) is only detectable in TET3c transformants with a DNA glycosylase mutant background suggesting further oxidation of 5 hmC residues to 5 fC by TET3c, and efficient recognition and removal of 5 fC by plant glycosylases. The results suggest that TET3c can be employed to induce heritable locus-specific changes in DNA methylation, and that accumulation of 5 hmC can be used as a marker for TET3c target regions.展开更多
Cell-free expression systems have emerged as a versatile and powerful platform for metabolic engineering,biosynthesis and synthetic biology studies.Nevertheless,successful examples of the synthesis of complex natural ...Cell-free expression systems have emerged as a versatile and powerful platform for metabolic engineering,biosynthesis and synthetic biology studies.Nevertheless,successful examples of the synthesis of complex natural products using this system are still limited.Bicyclomycin,a structurally unique and complex diketopiperazine alkaloid,is a clinically promising antibiotic that selectively inhibits the transcription termination factor Rho.Here,we established a modular cell-free expression system with cascade catalysis for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide.The six cell-free expressed biosynthetic enzymes,including five iron-andα-ketoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase,were active in converting their substrates to the corresponding products.The co-expressed enzymes in the cell-free module were able to complete the related partial pathway.In vitro biosynthesis of bicyclomycin was also achieved by reconstituting the entire biosynthetic pathways(i.e.,six enzymes)using the modular cell-free expression system.This study demonstrates that the modular cell-free expression system can be used as a robust and promising platformforthe biosynthesis of complex antibiotics.展开更多
We reported the characterization of a novel brassicicene diterpene biosynthetic gene cluster,which contains a uniqueα-ketoglutarate-dependent dioxygenase(αKGD)enzyme,AbnI.Our findings revealed that AbnI demonstrates...We reported the characterization of a novel brassicicene diterpene biosynthetic gene cluster,which contains a uniqueα-ketoglutarate-dependent dioxygenase(αKGD)enzyme,AbnI.Our findings revealed that AbnI demonstrates remarkable substrate promiscuity and is capable of activating multiple sites on both 5-8-5 and 5-9-5 brassicicene skeletons,resulting in skeleton modifications and an unexpected ring system rearrangement.These results suggested the potential utility of AbnI as an enzymatic tool for terpene C-H functionalization.In addition,the catalytic mechanism of AbnI and its potential ecological implications were discussed.展开更多
The dioxygen activation catalyzed by 4-hydorxylphenyl pyruvate dioxygenase(HPPD)were reinvestigated by using hybrid quantum mechanics/molecular mechanics(QM/MM)approaches at the B3LYP/6-311++G(d,p):AMBER level.These s...The dioxygen activation catalyzed by 4-hydorxylphenyl pyruvate dioxygenase(HPPD)were reinvestigated by using hybrid quantum mechanics/molecular mechanics(QM/MM)approaches at the B3LYP/6-311++G(d,p):AMBER level.These studies showed that this reaction consisted of two steps including the dioxygen addition/decarboxylation and hetero O-O bond cleavage,where the first step was found to be rate-determining.The former step initially runs on a septet potential energy surface(PES),then switches to a quintet PES after crossing a septet/quintet minimum energy crossing point(MECP)5-7M2,whereas the rest step runs on the quintet PES.The reliability of our theoretical predictions is supported by the excellent agreement of the calculated free-energy barrier value of 16.9 kcal/mol with available experimental value of 16-17 kcal/mol.The present study challenges the widely accepted view which holds that the O2activation catalyzed byα-keto glutamate(α-KG)dioxygenase mainly runs on the quintet PES and provides new insight into the catalytic mechanism ofα-KG dioxygenase and/or other related Fe(Ⅱ)-dependent oxygenase.展开更多
基金This work was supported by the National Key R&D Program of China(2020YFA0908000)the National Natural Science Foundation of China(81773830)+1 种基金the Key Project at central government level:The ability establishment of sustainable use for valuable Chinese medicine resources(2060302-1806-03)National Program for Special Support of Eminent Professionals.
文摘2-Oxoglutarate(2OG)-dependent dioxygenases(2-ODDs)are omnipresent iron-containing non-heme enzymes that catalyze various oxidation-reduction reactions in plant growth and development,nucleic acid modification and secondary metabolism.We systematically summarized recent research on the oxidative modifications of plant 2-ODDs and related enzymes,their vital importance in the biosynthesis of plant special metabolites,and their catalytic specificity/flexibility,and discussed the potential of 2-ODD as a new approach for the identification of pivotal genes and the elucidation of biosynthetic pathway.
基金the National Natural Science Foundation of China(no.21771087 to B.W and no.21703080 to J.C)the NSF of Shandong Province(no.ZR2020YQ10 to B.W)+1 种基金Taishan Scholar Program of Shandong Province(no.tsqn201812078 to B.W.)the NRF of Korea(no.NRF-2021R1A3B1076539 to W.N.and no.NRF-2020R1I1A1A01074630 to Y.-M.L.).
文摘Enantioselective cis-dihydroxylation of alkenes represents an ideal route to synthesize enantioenriched syn-2,3-dihydroxy esters that are important structural motifs in numerous biologically and pharmaceutically relevant molecules.Bioinspired nonheme iron-catalyzed enantioselective cis-dihydroxylation meets the requirement of the modern synthetic chemistry from the atomic economy,green chemistry,and sustainable development perspectives.However,nonheme iron-catalyzed enantioselective cis-dihydroxylation is much underdeveloped because of the formidable challenges of controlling chemo-and enantioselectivities and product selectivity caused by the competitive epoxidation,cis-dihydroxylation,and overoxidation reactions.Herein,we disclose the fabrication of a biologically inspired nonheme iron complex-catalyzed enantioselective cis-dihydroxylation of multisubstituted acrylates using hydrogen peroxide(H_(2)O_(2))as the terminal oxidant by controlling the non-ligating or weakly ligating counterions of iron(Ⅱ)complexes,demonstrating a dramatic counteranion effect on the enantioselective cisdihydroxylation of olefins by H_(2)O_(2) catalyzed by nonheme iron complexes.A range of structurally disparate alkenes were transformed to the corresponding syn-2,3-dihydroxy esters in practically useful yields with exquisite chemo-and enantioselectivities(up to 99% ee).Given the mild and benign nature of this biologically inspired oxidation system as well as the ubiquity and synthetic utility of enantioenriched syn-2,3-dihydroxy esters as pharmaceuticals candidates and natural products,we expect that this strategy could serve as a promising complement to the well-known Sharpless asymmetric dihydroxylation,which is the chemical reaction of an alkene with OsO_(4) to produce a vicinal diol.
基金financially supported by National Natural Science Foundation of China(Grant Nos.31961133030,31870678,32022076)Science Fund for Distinguished Young Scientists of Anhui Province(Grant No.1908085J12).
文摘The carotenoid-derived volatileβ-ionone makes an important contribution to tea fragrance.Here,we qualitatively and quantitatively analysed 15 carotenoids in tea leaves of 13 cultivars by UHPLC-APCI-MS/MS.The 13 cultivars were divided into two groups by PCA(Principal Component Analysis)clustering analysis of their carotenoid content,and OPLS-DA(Orthogonal projections to latent structures)indicated that the levels ofβ-carotene(VIP=2.89)and lutein(VIP=2.30)were responsible for much of the variation between the two groups.Interestingly,theβ-carotene toβ-ionone conversion rates in Group 1 were higher than in Group 2,while theβ-carotene content was significantly lower in Group 1 than in Group 2.Theβ-ionone content was significantly higher in Group 1.Pearson Correlation Coefficient calculation between the transcription level of candidate genes(CsCCD1 and CsCCD4)and the accumulation ofβ-ionone indicated that CsCCD1 may involve in the formation ofβ-ionone in 13 cultivars.Prokaryotic expression and in vitro enzyme activity assays showed that‘Chuanhuang 1’had an amino acid mutation in carotenoid cleavage dioxygenases 1(CsCCD1)compared with‘Shuchazao’,resulting in a significantly higherβ-ionone content in‘Chuanhuang 1’.Sequence analysis showed that‘Chuanhuang 1’and‘Huangdan’had different CsCCD1 promoter sequences,leading to significantly higher CsCCD1 expression andβ-ionone accumulation in‘Chuanhuang 1’.These results indicated that the promoter and coding sequence diversity of CsCCD1 might contribute to the differential accumulation ofβ-ionone in different tea cultivars.
基金Supported by the Postdoctoral Scientific Research Development Fund of Heilongjiang Province,China(LBH-Z21119)the Natural Science Fund Joint Guidance Project of Heilongjiang Province(LH2020C009)Young Talent Project of Northeast Agricultural University(19QC06)。
文摘Abscisic acid(ABA)is a plant hormone that plays an important role in plant development and abiotic stress.The 9-cis-epoxycarotenoid dioxygenase(NCED)is a key rate-limiting enzyme in the ABA biosynthetic pathway.The physiological and molecular mechanisms of NCED regulating plant development and abiotic stress tolerance have been reported in many plant species,but gene function of RiNCEDs in Rubus idaeus L.is rarely reported.In this study,the open reading frame(ORF)sequence of RiNCED2 in red raspberry fruit was isolated and the function of this gene under abiotic stress was investigated.While RiNCED2 was induced by cold,high salinity,drought and ABA,it was highly expressed in new leaves as measured by real-time qPCR.Overexpression of RiNCED2 in Arabidopsis under both high-salt and cold stress increased ABA content,demonstrating that RiNCED2 was involved in ABA biosynthesis.Meanwhile,the leaf wilting degree of transgenic Arabidopsis was less,while the content of malondialdehyde(MDA)was significantly reduced,and the chlorophyll content,proline content,peroxidase(POD),catalase(CAT)and superoxide dismutase(SOD)activities were significantly increased.These results indicated that overexpression of RiNCED2 enhanced the resistance of transgenic Arabidopsis to high salt and cold.
基金Supported by the National Natural Science Foundation of China (No.31271419 and No.31300329)
文摘Carotenoid cleavage dioxygenases(CCDs) are a class of enzymes in plants involved in the biosynthesis of apocarotenoids,such as phytohormones,flavour compounds,and other compounds with yet unknown functions.To date,several CCDs have been functionally characterised in plants,but little is known about the CCD4 members.A carotenoid cleavage dioxygenase 4 gene(LcCCD4) was isolated from the leaves of wolfberry(Lycium chinense) to gain insight into its biological function.Multiple sequence alignment and phylogenetic analyses showed that the deduced amino acid sequence of LcCCD4 shares high homology with that of CCD4 proteins from other plants.Expression analysis using semi-quantitative polymerase chain reaction revealed that LcCCD4 was strongly expressed in leaves and flowers and that the expression level was in accordance with β-carotene concentration.LcCCD4 transcripts in fruits tended to decrease as carotenoids accumulated.Recombinant expression of LcCCD4 cleaved β-carotene to produce P-ionone in in vivo assays.These results show that LcCCD4 is a CCD gene that may be involved in producing aromatic apocarotenoids in leaves and flowers,whereas it may be involved in controlling carotenoid accumulation in fruits.
基金supported by the National Natural Science Foundation of China (No. 50608011)the 39th Postdoctoral Funds of China.
文摘A novel biphenyl-degrading bacterial strain LA-4 was isolated from activated sludge. It was identified as Dyella ginsengisoli according to phylogenetic similarity of 16S rRNA gene sequence. This isolate could utilize biphenyl as sole source of carbon and energy, which degraded over 95 mg/L biphenyl within 36 h. The major metabolites formed from biphenyl, such as 2-hydroxy-6-oxo-6- phenylhexa-2,4-dienoic acid (HOPDA) and benzoic acid, were identified by LC-MS. The crude cell extract of strain LA-4 exhibited the activity of 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD) and the kinetic parameters Km was 26.48 μmol/L and Vmax was 8.12 U/mg protein. A conserved region of the biphenyl dioxygenase gene bphA1 of strain LA-4 was amplified by PCR and confirmed by DNA sequencing.
基金Science and Technology Program of Gansu Province Grant,No.18JR3RA339 and No.18JR3RA363Fund of the First Hospital of Lanzhou University Grant,No.ldyyyn2018-63+3 种基金Teaching and Research Project of the First Clinical Medical College of Lanzhou University in 2018 Grant,No.2018007NIH awards,No.R21NS106430 and No.R01OD026594Cystic Fibrosis Foundation Research Grant,No.ZHAO19G0an American Cancer Society-IRG Junior Faculty Development Grant,a UAB CCC Neuro-oncology Research Acceleration Grant,and a UAB Faculty Development Grant Program Award.
文摘Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors of the gastrointestinal tract.At the molecular level,GISTs can be categorized into two groups based on the causative oncogenic mutations.Approximately 85%of GISTs are caused by gain-of-function mutations in the tyrosine kinase receptor KIT or platelet-derived growth factor receptor alpha(PDGFRA).The remaining GISTs,referred to as wild-type(WT)GISTs,are often deficient in succinate dehydrogenase complex(SDH),a key metabolic enzyme complex in the tricarboxylic acid(TCA)cycle and electron transport chain.SDH deficiency leads to the accumulation of succinate,a metabolite produced by the TCA cycle.Succinate inhibitsα-ketoglutarate-dependent dioxygenase family enzymes,which comprise approximately 60 members and regulate key aspects of tumorigenesis such as DNA and histone demethylation,hypoxia responses,and m6A mRNA modification.For this reason,succinate and metabolites with similar structures,such as D-2-hydroxyglutarate and fumarate,are considered oncometabolites.In this article,we review recent advances in the understanding of how metabolic enzyme mutations and oncometabolites drive human cancer with an emphasis on SDH mutations and succinate in WT GISTs.
基金supported by grants from the National Natural Science Foundation of China(GrantNo.31271311)the Ministry of Agriculture of China(Grant No.2011ZX08009-003)
文摘High-tillering dwarf mutant gsor23 was generated from an indica rice variety Indica9 radiatied by y-ray. Genetic analysis showed that this phenotype was controlled by one single recessive gene, which was mapped within a physical distance of 386 kb between two insertion-deletion (InDel) markers CI-WT2 and C1-WT4 on the long arm of chromosome 1. There is a known gene DIO within this region, the mutation of which causes high-tillering in rice. Sequence analysis of the DIO allele in gsor23 revealed that the base cytosine (C) at the 404th position in the coding region was deleted, which would cause frameshift mutation after the 134th amino acids. The mutation site and indica background of gsor23 were different from the previously reported japonica mutants d10-1 and d10-2. Therefore, gsor23 is a novel allelic mutant of D10 which encodes the carotenoid-cleaving dioxygenase 8 (CCD8), a key enzyme involved in the biosynthesis of the new plant hormone strigolactones (SLs). After treatment with GR24, a synthetic analogue of SLs, the high-tillering phenotype of gsor23 was restored to normal. Real-time RT-PCR analysis showed that D10 expression was high in roots, but low in leaves. Compared with the wild type Indica9, the expression of the SL biosynthesis gene DIO was upregulated, while genes likely involved in the SL signal transduction pathway such as D3 and D14 were down-regulated in the gsor23 mutant.
基金Supported by DSR Scholarship Support,King Saud University。
文摘Lutein,a type of carotenoids,is found to delay the onset and progression of age-related macular degeneration(AMD).Several lutein supplementation studies showed that after an initial increase,lutein serum levels demonstrated a subsequent decrease despite continuous supplementation.In this systematic literature review,this obscure phenomenon was tried to be explained.The subsequent drop in lutein levels was postulated due to down-regulation of lutein receptors scavenger receptor class B typeⅠ(SR-BI)in the gastrointestinal tract,upregulation of lutein degrading enzymeβ-carotene dioxygenase(BCDO2),or perhaps a combination of both.Some single nucleotides polymorphisms(SNPs)that could have influence on the occurrence of this phenomenon.To date,an exact scientific explanation for this phenomenon has not been established.Further research is needed to investigate this phenomenon in depth to reach an irrefutable explanation,giving that lutein is proven to be effective in delaying the onset and progression of AMD and its metabolism in the human body becomes of equal importance.
基金Supported by the National Natural Science Foundation of China(Nos.41101226,50879029)the Technology Development Project of Jilin Province,China(Nos.201101020,20090415)
文摘To find new extradiol dioxygenases(EDOs,EC 1.13.11.2),a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity.A novel EDO,designated as BphC_A,was identified and heterologously expressed in Escherichia coli.The deduced amino acid sequence of BphC_A exhibited a homology of less than 60% with other known EDOs.Phylogenetic analysis of BphC_A suggests that the protein is a novel member of the EDO family.The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol,the preferred substrate of other known EDOs.The optimum activity of purified BphC_A occurred at pH=8.5 and 35 °C,and BphC_A showed more than 40% of its initial activity at 5 °C.The activity of purified BphC_A was significantly induced by Mn^2+ and slightly reduced by Al^3+,Cu^2+ and Zn^2+.
文摘The Pseudomonas putida TOL plasmid gene xy1E,whichproduces catechol 2,3-dioxygenase (CatO<sub>2</sub>ase;catechol:oxygen 2,3-oxidoreductase (decyclizing),EC 1.13.11.2 )was used as a target gene to study the mechanisms ofmutation and induced mutation frequency in
文摘To construct a high-level expression of xylE gene in E. coli for quick detection of environmental pollution of aromatic compounds. Methods and Results: XylE gene coding for the catechol 2, 3 dioxygenase (CatO2ase ) was amplified from the recombinant plasmid pTG402 by using PCR technique and was subcloned into pUC118N and pUC119N. The single stranded recombinant phage DNA from the transformed E. coli MV1184 cells was used for sequencing. The sequence of xylE gene was proved to be the same as reported. The gene was then subcloned into the high expression plasmid pJLA503, its expression amount being about 34. 2% of the total bacterial proteins. Conclusion: xylE gene is highly expressed in host E. coli TG1.
文摘In plants, demethylation of 5-methylcytosine (5 mC) residues is controlled by DNA glycosylases, while in mammals it requires oxidation of 5 mC by TET proteins, a group of Fe(II)/2-oxoglutaratedependent dioxygenases. We analysed the effects of expressing the C-terminal catalytic domain of the human TET3 gene (TET3c) in Arabidopsis thaliana, using an rDNA region as a methylation reporter. In TET3c transformants, epialleles with hypomethylation or hypermethylation patterns can be induced, which is each stably retained in progeny lines even after removal of the TET3c transgene. In TET3c transformants, 5-hydroxymethylcytosine (5 hmC) marks are detected, indicative of the oxidative activity of the transgenic enzyme. 5-formylcytosine (5 fC) is only detectable in TET3c transformants with a DNA glycosylase mutant background suggesting further oxidation of 5 hmC residues to 5 fC by TET3c, and efficient recognition and removal of 5 fC by plant glycosylases. The results suggest that TET3c can be employed to induce heritable locus-specific changes in DNA methylation, and that accumulation of 5 hmC can be used as a marker for TET3c target regions.
基金supported in part by grants from the National Key Research and Development Program of China(2022YFC2303100)the National Natural Science Foundation of China(22207117).
文摘Cell-free expression systems have emerged as a versatile and powerful platform for metabolic engineering,biosynthesis and synthetic biology studies.Nevertheless,successful examples of the synthesis of complex natural products using this system are still limited.Bicyclomycin,a structurally unique and complex diketopiperazine alkaloid,is a clinically promising antibiotic that selectively inhibits the transcription termination factor Rho.Here,we established a modular cell-free expression system with cascade catalysis for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide.The six cell-free expressed biosynthetic enzymes,including five iron-andα-ketoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase,were active in converting their substrates to the corresponding products.The co-expressed enzymes in the cell-free module were able to complete the related partial pathway.In vitro biosynthesis of bicyclomycin was also achieved by reconstituting the entire biosynthetic pathways(i.e.,six enzymes)using the modular cell-free expression system.This study demonstrates that the modular cell-free expression system can be used as a robust and promising platformforthe biosynthesis of complex antibiotics.
基金financially supported by the National Key R&D Program of China(Nos.2021YFA0910500,2021YFA0910503)the National Natural Science Foundation of China(Nos.22277035,32000045 and 81973205)+4 种基金the Program for Changjiang Scholars of Ministry of Education of the People’s Republic of China(No.T2016088)the National Natural Science Foundation for Distinguished Young Scholars(No.81725021)the National Science and Technology Project of China(No.2018ZX09201001-001-003)Innovative Research Groups of the National Natural Science Foundation of China(No.81721005)the Fundamental Research Funds for the Central Universities(No.2020kfyXJJS043).
文摘We reported the characterization of a novel brassicicene diterpene biosynthetic gene cluster,which contains a uniqueα-ketoglutarate-dependent dioxygenase(αKGD)enzyme,AbnI.Our findings revealed that AbnI demonstrates remarkable substrate promiscuity and is capable of activating multiple sites on both 5-8-5 and 5-9-5 brassicicene skeletons,resulting in skeleton modifications and an unexpected ring system rearrangement.These results suggested the potential utility of AbnI as an enzymatic tool for terpene C-H functionalization.In addition,the catalytic mechanism of AbnI and its potential ecological implications were discussed.
基金supported by the National Key R&D Program(No.2021YFD1700100)National Natural Science Foundation of China(Nos.21837001,21273089)+2 种基金the Open Project Fund of the Key Laboratory of the Pesticides and Chemical Biology of Central China Normal University(No.2018-A01)the Fundamental Research Funds for the Central Universitiesthe Fundamental Research Funds for the South-Central University for Nationalities(No.CZW20020)。
文摘The dioxygen activation catalyzed by 4-hydorxylphenyl pyruvate dioxygenase(HPPD)were reinvestigated by using hybrid quantum mechanics/molecular mechanics(QM/MM)approaches at the B3LYP/6-311++G(d,p):AMBER level.These studies showed that this reaction consisted of two steps including the dioxygen addition/decarboxylation and hetero O-O bond cleavage,where the first step was found to be rate-determining.The former step initially runs on a septet potential energy surface(PES),then switches to a quintet PES after crossing a septet/quintet minimum energy crossing point(MECP)5-7M2,whereas the rest step runs on the quintet PES.The reliability of our theoretical predictions is supported by the excellent agreement of the calculated free-energy barrier value of 16.9 kcal/mol with available experimental value of 16-17 kcal/mol.The present study challenges the widely accepted view which holds that the O2activation catalyzed byα-keto glutamate(α-KG)dioxygenase mainly runs on the quintet PES and provides new insight into the catalytic mechanism ofα-KG dioxygenase and/or other related Fe(Ⅱ)-dependent oxygenase.