拟南芥Alpha-dioxygenase1的原核表达、纯化及活性的检测

从经水杨酸处理的拟南芥开花期植株中获得cDNA,扩增得到Alpha-dioxygenase 1(DOX1)基因,进行原核表达、纯化和生物活性检测。利用原核表达载体pMAL-c4x在T7 Express CompetentE.coli和BL21(DE3)-RIPL codon+菌株中表达DOX1,经Amylose Resin亲和层析柱纯化。SDS-PAGE结果表明,重组融合蛋白在BL21(DE3)-RIPL codon+中的表达通过灰度值比较分析以可溶性为主,表达率约3.7%,纯化的DOX1纯度可达45%。愈创木酚法表明,可溶性重组蛋白不具有过氧化物酶活性;2,4-DNP法测试表明可溶性重组蛋白具有脂肪酸双加氧酶活性。

Structural insights of 4-Hydrophenylpyruvate dioxygenase inhibition by structurally diverse small molecules

4-Hydrophenylpyruvate dioxygenase(HPPD),a key enzyme involved in tyrosine catabolism,has long been considered a promising target for herbicides and drugs.Several types of HPPD inhibitors have been developed as high-potency drugs or herbicides.Understanding the structural basis of the binding of these inhibitors with HPPD will be beneficial for the development of inhibitors containing novel scaffolds.However,only limited structural information regarding the binding of triketone and pyrazole derivatives with HPPD has been reported.Here,the crystal structures of HPPD complexed with inhibitors containing different scaffolds,including triketone,pyrazole,isoxazole,heterocyclic amide,and quinazolindione derivatives,were comprehensively analyzed.Detailed binding modes of isoxazole and heterocyclic amide derivatives with HPPD were first revealed,facilitating further structural optimization.The binding mode of compound 2 with HPPD suggests that both oxygen and nitrogen atoms can mediate coordination with the metal ion.These results will provide the structure-based rational design of novel HPPD inhibitors.

Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil

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＋.

Subcloning and high level expression of xylE gene coding for the catechol 2, 3 dioxygenase in E. coli

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.

Regulation of A 9-cis-epoxycarotenoid Dioxygenase(NCED)Gene from Raspberry on High Salinity and Cold Tolerance

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.

Transcriptional Analysis of 9-Cis-Epoxycarotenoid Dioxygenase, Glucosyltransferase, 8＇-Hydroxylase and B-Glucosidase Genes that Regulate Abscisic Acid Homeostasis around the Onset of Grape Berry Ripening

The mechanisms for fine-tuning ABA level related to grape berry ripening remain elusive. Here, transcription analysis showed that the mRNA expression level of 9-cis-epoxycarotenoid dioxygenase gene （VvNCED1） increases first, rapidly in mesocarp before the onset of grape berry ripening. After VvNCED1 peaks its expression level, ABA content increases rapidly in mesocarp coupled with an increase in both soluble sugar content and pH value. On the onset of berry ripening, VvNCED1 transcripts decline rapidly to its lowest point, then increases slightly. Whereas, the mRNA expression level of B-glucosidase gene VvBGI, on the whole, increases constantly during grape berry ripening. During berry de-greening, ABA glucosyltransferase （VvGT） and ABA 8＇-hydroxylase （VvCYPI） equilibrate ABA level; during berry coloring-up, VvGT predominantly equilibrates ABA level, namely, the up-regulation of ABA level mainly leads from VvNCED1 and VvBG1 gene high expression; the down-regulation of ABA level leads mainly from VvCYP! transcript level both in ABA content- and developmental phase-dependence manner. In conclusion, our main results show that VvNCED1 and VvBG1 genes are closely related to grape berry ripening.

2-Oxoglutarate-dependent dioxygenases in plants:biosynthesis,mechanism and evolution

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.

Expression of the C-Terminal Domain of Mammalian TET3 DNA Dioxygenase in Arabidopsis thaliana Induces Heritable Methylation Changes at rDNA Loci

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.

AbnI:Anα-ketoglutarate-dependent dioxygenase involved in brassicicene C-H functionalization and ring system rearrangement

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.

A newly defined dioxygenase system from Mycobacterium vanbaalenii PYR-1 endowed with an enhanced activity of dihydroxylation of high-molecular-weight polyaromatic hydrocarbons

NidA3B3 is a terminal dioxygenase whose favorable substrates are high-molecular-weight polyaromatic hydrocarbons(PAHs)from Mycobacterium vanbaalenii PYR-1,a powerftil PAHs degradation strain.NidA3B3 was reported to incorporate a dioxygen into the benzene ring of PAHs when equipped with an exogenous electron transport chain components PhdCD from Nocarciioides sp.strain KP7 by biotransformation,but this enzyme system was not particularly efficient.In this study,strain PYR-1 was confirmed to utilize four different PAHs at different growth rates.When PhtAcAd,an endogenous electron transport chain of a phthalate dioxygenase system,was substituted for PhdCD to couple with NidA3B3,the specific activity to convert phenanthrene by strain BL21(DE3)[pNidA3B3-PhAcAd]was 0.15±0.03 U/mg,but the specific activity of strain BL21(DE3)[pNidA3B3-PhdCD]was only 0.025±0.006U/mg.In addition,FNidA3,encoded by a newly defined ORF,has a prolonged 19-amino acid sequence at the N-terminus compared with NidA3.FNidA3B3 increased the activity by 50%approximately than NidA3B3 when using PhtAcAd.Components of the electron transport chain PhtAc and PhtAd were purified and characterized.The Km,kcal,kcat/Km values of the PhtAd were 123±26.9 pM,503±49.9 min^-1,4.1μM^-1·min^-1,respectively.And the,Km,Kcat,Kcat/Km values of the ferredoxin PhtAc were (52.5±9.7)μM,3.8±0.19min^-1 and 0.07μM^-1 min1,respectively.Basing on the phylogenetic analysis,NidA3/FNidA3 were far from its isoenzyme NidA from the same strain.Combining their primary differences of transcriptional pattern in vivo,it indicated that the functionally similar Rieske dioxygenases NidA3B3/FNidA3B3 and NidAB might originate from different ancestors.

Effect of salinity on community structure and naphthalene dioxygenase gene diversity of a halophilic bacterial consortium

The aim of this study is to analyze the effect of salinity on polycyclic aromatic hydrocarbons （PAHs） biodegradation, community structure and naphthalene dioxygenase gene （ndo） diversity of a halophilic bacterial consortium with the denaturing gradient gel electrophoresis （DGGE） approach. The consortium was developed from oil-contaminated saline soil after enrichment for six times, using phenanthrene as the substrate. The prominent species in the bacterial consortium at all salinities were identified as halophilic bacteria Halomonas, Alcanivorax, Marinobacter, Idiomarina, Martelella and uncultured bacteria. The predominant microbes gradually changed associating with the saline concentration fluctuations ranging from 0.1% to 25% （w/v）. Two ndo alpha subunits were dominant at salinities ranging, from 0.1% to 20%. while not been clearly detected at 25% salinity. Consistently. the biodegradation occurred at salmltles rangmg from 0.1% to 20%, while no at 25% salinity, suggesting the two ndo genes played an important role in the degradation. The phylogenetic analysis revealed that both of the two ndo alpha subunits were related to the classic nab-like gene from Pseudomonas stutzeri AN10 and Pseudomonas aeruginosa PaK1, while one with identity of about 82% and the other one with identity of 90% at amino acid sequence level. We concluded that salinity greatly affected halophilic bacterial community structure and also the functional genes which were more related to biodegradation.

The structure of 4-hydroxylphenylpyruvate dioxygenase complexed with 4-hydroxylphenylpyruvic acid reveals an unexpected inhibition mechanism

4-Hydroxyphenylpyruvate dioxygenase(HPPD)is an important target for both drug and pesticide discovery.As a typical Fe(II)-dependent dioxygenase,HPPD catalyzes the complicated transformation of 4-hydroxyphenylpyruvic acid(HPPA)to homogentisic acid(HGA).The binding mode of HPPA in the catalytic pocket of HPPD is a focus of research interests.Recently,we reported the crystal structure of Arabidopsis thaliana HPPD(At HPPD)complexed with HPPA and a cobalt ion,which was supposed to mimic the pre-reactive structure of At HPPD-HPPA-Fe(II).Unexpectedly,the present study shows that the restored At HPPD-HPPA-Fe(II)complex is still nonreactive toward the bound dioxygen.QM/MM and QM calculations reveal that the HPPA resists the electrophilic attacking of the bound dioxygen by the trim of its phenyl ring,and the residue Phe381 plays a key role in orienting the phenyl ring.Kinetic study on the F381 A mutant reveals that the HPPD-HPPA complex observed in the crystal structure should be an intermediate of the substrate transportation instead of the pre-reactive complex.More importantly,the binding mode of the HPPA in this complex is shared with several well-known HPPD inhibitors,suggesting that these inhibitors resist the association of dioxygen(and exert their inhibitory roles)in the same way as the HPPA.The present study provides insights into the inhibition mechanism of HPPD inhibitors.

Advances in Fe(II)/2-ketoglutarate-dependent dioxygenase-mediated C-H bond oxidation for regioselective and stereoselective hydroxyl amino acid synthesis:from structural insights into practical applications

The asymmetric hydroxylation of inactive carbon atoms in organic compounds remains an important reaction in the industrial synthesis of valuable chiral compounds.Fe(II)and 2-ketoglutarate-dependent dioxygenases(Fe/2-kg DOs)are the largest known subgroups of mononuclear nonheme-Fe(II)-dependent oxygenases,catalyzing various oxidation reactions of C-H bonds.Recent developments in Fe/2-kg DO-related researches have coupled concepts from bioinformatics,synthetic biology,and computational biology to establish effective biotransformation systems.The most well-studied and characterized activ-ity of the Fe/2-kg DOs is substrate hydroxylation,with regard to which mechanistic studies involving the Fe center assist in engineering the protein frameworks of these enzymes to obtain the desired catalytic enhancements.Amino acids are typical substrates of Fe/2-kg DOs and are usually converted into hydroxyl amino acids,which are widely used as intermediates in pharmaceutical and fine chemical industries.Herein,we have reviewed prior structural and mechanistic studies on Fe/2-kg DOs,as well as studies on the Fe/2-kg DO-mediated selective C-H oxidation process for selective hydroxyl amino acid synthesis,which will further our journey along the promising path of building complexity via C-H bond oxidation.Further,new bioinformatics techniques should be adopted with structure-based protein rational design to mine sequence databases and shrink mutant libraries to produce a diverse panel of functional Fe/2-kg DOs capable of catalyzing targeted reactions.

Analysis of Affinity Energy Between Biphenyl Dioxygenase and Polychlorinated Biphenyls Using Molecular Docking

Molecular docking was used to calculate the affinity energy between biphenyl dioxygenases(BphA),including 1ULJ,1WQL,2YFJ,2YFL,2GBX,2XSH,2B4P,3GZX,and 3GZY(selected from the Protein Data Bank)and 209 polychlorinated biphenyl(PCB)congeners.The relationships between the calculated affinity energy and the persistent organic pollutant characteristics(migration,octanol-air partition coefficients,lgKOA;persistence,half-life,lgt1/2;toxicity,half-maximal inhibitory concentration,IgIC50;bioaccumulation,bioconcentration factor,lgBCF)of the PCBs were studied to mderstand the BphA mediated degradation of PCBs.The effect of substituent characteristics on the affinity energy was explored through full factorial experimental design.The affinities of nine kinds of BphA proteins on PCBs ranked as follows:2GBX>2YFJ>2YFL>3GZX>2XSH>3GZY>2E4P>1 WQL>1ULJ.The relationships between the calculated affinity energy and the molecular weight,lgKOA,lgBCF,and lgt1/2 of the PCBs were statistically signiflcant(p<0.01),whereas the relationship with the lgIC50 of PCBs was not statistically significant(p>0.05).PCBs were more difficult to degrade following an increase in the free energy of binding.Correlation analysis showed that the average affinity energy values of PCBs gradually increased as the number of chlorine atoms increased,regardless of the substituent position.The substituents at the ortho-positions interacted mainly through a second-order interaction,whereas those at the para-positions did not participate via a second-order interaction.

QM/MM study on the O_(2)activation reaction of 4-hydroxylphenyl pyruvate dioxygenase reveals a common mechanism forα-ketoglutarate dependent dioxygenase

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.

The dioxygenase GIM_2 functions in seed germination by altering gibberellin production in Arabidopsis

The phytohormones gibberellic acid （GA） and abscisic acid （ABA） antagonistically control seed germina- tion. High levels of GA favor seed germination, whereas high levels of ABA hinder this process. The direct relationship between GA biosynthesis and seed germina- tion ability need further investigation. Here, we identified the ABA-insensitive gain-of-function mutant germination insensitive to ABA mutant 2 （gim2） by screening a population of XVE T-DNA-tagged mutant lines. Based on two loss-of-function gim2-ko mutant lines, the disruption of GIM2 function caused a delay in seed germination. By contrast, upregulation of GIM2 accelerated seed germina- tion, as observed in transgenic lines overexpressing GIM2 （OE）. We detected a reduction in endogenous bioactive GA levels and an increase in endogenous ABA levels in the gim2-ko mutants compared to wild type. Conversely, the OE lines had increased endogenous bioactive GA levels and decreased endogenous ABA levels. The expression levels of a set of GA- and]or ABA-related genes were altered in both the gim2-ko mutants and the OE lines. We confirmed that GIM2 has dioxygenase activity using an in vitro enzyme assay, observing that GIM2 can oxidize GA^2. Hence, our characterization of GIM2 demonstrates that it plays a role in seed germination by affecting the GA metabolic pathway in Arabidopsis.

Nonheme Iron-Catalyzed Enantioselective cis-Dihydroxylation of Aliphatic Acrylates as Mimics of Rieske Dioxygenases

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.

用于哺乳动物细胞突变分析的EBV-xyIE穿梭质粒系统的建立

以恶臭假单孢菌(Pseudonas putida)TOL质粒来的xylE基因为靶因可方便地进行突变分析,该基因编码邻苯二酚2,3-双加氧酶(Catechol 2,3-dioxygenase),或称邻苯二酚化酶(CatOz ase),通过喷洒邻苯二酚于氨苄青霉素抗性(ampr)菌落上,xylE-转化子就能检测出来,菌落变黄表示存在邻苯二酚氧化酶。我们在以前构建的EBV-xylE穿梭质粒pFD891(王晓利等,复旦学报理科版,1990第4期)

Immunotherapy and immunoescape in colorectal cancer

Immunotherapy encompasses a variety of interventions and techniques with the common goal of eliciting tumor cell destructive immune responses. Colorectal carcinoma often presents as metastatic disease that impedes curative surgery. Novel strategies such as active immunization with dendritic cells (DCs), gene transfer of cytokines into tumor cells or administration of immunostimulatory monoclonal antibodies (such as anti-CD137 or anti-CTLA-4) have been assessed in preclinical studies and are at an early clinical development stage. Importantly, there is accumulating evidence that chemotherapy and immunotherapy can be combined in the treatment of some cases with colorectal cancer, with synergistic potentiation as a result of antigens cross-presented by dendritic cells and/or elimination of competitor or suppressive T lymphocyte populations (regulatory T-cells). However, genetic and epigenetic unstable carcinoma cells frequently evolve mechanisms of immunoevasion that are the result of either loss of antigen presentation, or an active expression of immunosuppressive substances. Some of these actively immunosuppressive mechanisms are inducible by cytokines that signify the arrival of an effector immune response. For example, induction of 2, 3 indoleamine dioxygenase (IDO) by IFNγ in colorectal carcinoma cells. Combinational and balanced strategies fostering antigen presentation, T-cell costimulation and interference with immune regulatory mechanisms will probably take the stage in translational research in the treatment of colorectal carcinoma.