An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and ...An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.展开更多
In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL 1 was constructed. The recombinant was identified by three restriction endonucleases, then...In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL 1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL 1 was transformed into expression host M15 （pREP4） and induced by isopropyl-a-D-thiogalactoside （IPTG） to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol＇L-1 IPTG induction as the expression temperature declined from 37 to 28℃. The 6-His tag facilitates affinity binding to Ni^2＋-nitrolotriacetic acid （NTA） and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose coupled with Ni^2＋-NTA affinity chromatography. The optimal substrate for Pt4CL 1 was 4-coumarate.展开更多
In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1（SIAH-1） in PC12 cells. 1-Methyl-4-phenylpyridinium（MPP＋） treatment increased α-synuclein, E1 and S...In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1（SIAH-1） in PC12 cells. 1-Methyl-4-phenylpyridinium（MPP＋） treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased m RNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 m RNA expression. It also increased cell viability. Combined treatment with MPP＋ and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson’s disease.展开更多
4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the me...4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen （Salvia miltiorrhiza Bunge）, a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs （Sm4CL1 and Sm4CL2） encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction （RT-PCR） with degenerate primers followed by 5′/3′rapid amplification of cDNA ends （RACE） （Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164）. Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were （72.20±4.10） and （6.50±1.45） μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.展开更多
According to synthetic pathway of plant chlorogenic acid （CGA）, the expression patterns of genes encoding enzymes that are associated with CGA synthesis were studied in normally developed Xuehua pear fruit. The stud...According to synthetic pathway of plant chlorogenic acid （CGA）, the expression patterns of genes encoding enzymes that are associated with CGA synthesis were studied in normally developed Xuehua pear fruit. The study demonstrated that CGA content in peel and flesh of Xuehua pear decreased as fruit development progressed, with a higher level in peel. The expression levels of PbPAL 1, PbPAL2, PbC3H, PbC4H, Pb4CL 1, Pb4CL2, Pb4CL6, PbHC T1 and PbHC T3 genes decreased in fruit, which was consistent with the pattern of variation in CGA content. That indicated that these genes might be key genes for influencing fruit CGA synthesis in Xuehua pear. However, Pb4CL7 gene expression profile is not consistent with variation of CGA content, hence, it may not be a key gene involved in CGA synthesis.展开更多
DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes.Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end ...DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes.Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end joining(NHEJ)system and ultimately cause genome mutagenesis.Here,we demonstrated that Enterobacteria phage T4 DNA ligase alone is capable of mediating in vivo chromosome DSBs repair in Escherichia coli.The ligation efficiency of DSBs with T4 DNA ligase is one order of magnitude higher than the NHEJ system from Mycobacterium tuberculosis.This process introduces chromosome DNA excision with different sizes,which can be manipulated by regulating the activity of host-exonuclease RecBCD.The DNA deletion length reduced either by inactivating recB or expressing the RecBCD inhibitor Gam protein fromλphage.Furthermore,we also found single nucleotide substitutions at the DNA junction,suggesting that T4 DNA ligase,as a single component non-homologous end joining system,has great potential in genome mutagenesis,genome reduction and genome editing.展开更多
The key enzymes involved in the flavonoid biosynthesis pathway have been extensively studied in seed plants,but relatively less in ferns.In this study,two 4-Coumarate:coenzyme A ligases(Sc4CL1 and Sc4CL2)and one novel...The key enzymes involved in the flavonoid biosynthesis pathway have been extensively studied in seed plants,but relatively less in ferns.In this study,two 4-Coumarate:coenzyme A ligases(Sc4CL1 and Sc4CL2)and one novel chalcone synthase(ScCHS1)were functionally characterized by mining the Stenoloma chusanum transcriptome database.Recombinant Sc4CLs were able to esterify various hydroxycinnamic acids to corresponding acyl-coenzyme A(CoA).ScCHS1could catalyze p-coumaroyl-CoA,cinnamoyl-CoA,caffeoyl-CoA,and feruloyl-CoA to form naringenin,pinocembrin,eriodictyol,and homoeriodictyol,respectively.Moreover,enzymatic kinetics studies revealed that the optimal substrates of ScCHS1were feruloyl-CoA and caffeoyl-CoA,rather than p-coumaroyl-CoA,which was substantially different from the common CHSs.Crystallographic and sitedirected mutagenesis experiments indicated that the amino acid residues,Leu87,Leu97,Met165,and Ile200,located in the substrate-binding pocket near the B-ring of products,could exert a significant impact on the unique catalytic activity of ScCHS1.Furthermore,overexpression of ScCHS1 in tt4 mutants could partially rescue the mutant phenotypes.Finally,ScCHS1 and Sc4CL1 were used to synthesize flavanones and flavones with multi-substituted hydroxyl and methoxyl B-ring in Escherichia coli,which can effectively eliminate the need for the cytochrome P450 hydroxylation/O-methyltransferase from simple phenylpropanoid acids.In summary,the identification of these important Stenoloma enzymes provides a springboard for the future production of various flavonoids in E.coli.展开更多
文摘An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.
文摘In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL 1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL 1 was transformed into expression host M15 （pREP4） and induced by isopropyl-a-D-thiogalactoside （IPTG） to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol＇L-1 IPTG induction as the expression temperature declined from 37 to 28℃. The 6-His tag facilitates affinity binding to Ni^2＋-nitrolotriacetic acid （NTA） and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose coupled with Ni^2＋-NTA affinity chromatography. The optimal substrate for Pt4CL 1 was 4-coumarate.
基金supported by the China Postdoctoral Science Foundation,No.1630the Natural Science Foundation of Jiangsu Province in China,No.BK2011402+1 种基金the Jiangsu Province Postdoctoral Research Foundation in China,No.1301174Cthe Jiangsu Province Health Department Foundation in China,No.H201361
文摘In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1（SIAH-1） in PC12 cells. 1-Methyl-4-phenylpyridinium（MPP＋） treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased m RNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 m RNA expression. It also increased cell viability. Combined treatment with MPP＋ and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson’s disease.
基金Supported by the National Natural Science Foundation of China （30300447）.The authors thank Dr Chen Yongning （China Innovation Centre for Drug Development, HK） for useful suggestions and support. The authors also thank to Dr Fanya Zeng and Miss Charis Chan （Department of Zoology, University of Hong Kong） for technical assistance.
文摘4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen （Salvia miltiorrhiza Bunge）, a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs （Sm4CL1 and Sm4CL2） encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction （RT-PCR） with degenerate primers followed by 5′/3′rapid amplification of cDNA ends （RACE） （Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164）. Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were （72.20±4.10） and （6.50±1.45） μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.
基金supported by the Specific Financial Funds of Hebei Province,China (494-0502-JSN-7FB3)
文摘According to synthetic pathway of plant chlorogenic acid （CGA）, the expression patterns of genes encoding enzymes that are associated with CGA synthesis were studied in normally developed Xuehua pear fruit. The study demonstrated that CGA content in peel and flesh of Xuehua pear decreased as fruit development progressed, with a higher level in peel. The expression levels of PbPAL 1, PbPAL2, PbC3H, PbC4H, Pb4CL 1, Pb4CL2, Pb4CL6, PbHC T1 and PbHC T3 genes decreased in fruit, which was consistent with the pattern of variation in CGA content. That indicated that these genes might be key genes for influencing fruit CGA synthesis in Xuehua pear. However, Pb4CL7 gene expression profile is not consistent with variation of CGA content, hence, it may not be a key gene involved in CGA synthesis.
基金This work was supported by grants from the National Natural Science Foundation of China[31730003,31670077]Natural Science Foundation of Shandong Province[ZR2017ZB0210].
文摘DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes.Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end joining(NHEJ)system and ultimately cause genome mutagenesis.Here,we demonstrated that Enterobacteria phage T4 DNA ligase alone is capable of mediating in vivo chromosome DSBs repair in Escherichia coli.The ligation efficiency of DSBs with T4 DNA ligase is one order of magnitude higher than the NHEJ system from Mycobacterium tuberculosis.This process introduces chromosome DNA excision with different sizes,which can be manipulated by regulating the activity of host-exonuclease RecBCD.The DNA deletion length reduced either by inactivating recB or expressing the RecBCD inhibitor Gam protein fromλphage.Furthermore,we also found single nucleotide substitutions at the DNA junction,suggesting that T4 DNA ligase,as a single component non-homologous end joining system,has great potential in genome mutagenesis,genome reduction and genome editing.
基金supported by the National Natural Science Foundation of China (No. 31770330 and 31870720)supported by the Foundation of Youth Innovation Promotion Association of the Chinese Academy of Sciences
文摘The key enzymes involved in the flavonoid biosynthesis pathway have been extensively studied in seed plants,but relatively less in ferns.In this study,two 4-Coumarate:coenzyme A ligases(Sc4CL1 and Sc4CL2)and one novel chalcone synthase(ScCHS1)were functionally characterized by mining the Stenoloma chusanum transcriptome database.Recombinant Sc4CLs were able to esterify various hydroxycinnamic acids to corresponding acyl-coenzyme A(CoA).ScCHS1could catalyze p-coumaroyl-CoA,cinnamoyl-CoA,caffeoyl-CoA,and feruloyl-CoA to form naringenin,pinocembrin,eriodictyol,and homoeriodictyol,respectively.Moreover,enzymatic kinetics studies revealed that the optimal substrates of ScCHS1were feruloyl-CoA and caffeoyl-CoA,rather than p-coumaroyl-CoA,which was substantially different from the common CHSs.Crystallographic and sitedirected mutagenesis experiments indicated that the amino acid residues,Leu87,Leu97,Met165,and Ile200,located in the substrate-binding pocket near the B-ring of products,could exert a significant impact on the unique catalytic activity of ScCHS1.Furthermore,overexpression of ScCHS1 in tt4 mutants could partially rescue the mutant phenotypes.Finally,ScCHS1 and Sc4CL1 were used to synthesize flavanones and flavones with multi-substituted hydroxyl and methoxyl B-ring in Escherichia coli,which can effectively eliminate the need for the cytochrome P450 hydroxylation/O-methyltransferase from simple phenylpropanoid acids.In summary,the identification of these important Stenoloma enzymes provides a springboard for the future production of various flavonoids in E.coli.
