The acetylpolyamine oxidase(APAO),spermine oxidase(SMO),and spermidine/spermine N1-acetyltransferase(SSAT)are pivotal enzymes in polyamine metabolism,exerting direct influence on polyamine homeostasis regulation.Dysfu...The acetylpolyamine oxidase(APAO),spermine oxidase(SMO),and spermidine/spermine N1-acetyltransferase(SSAT)are pivotal enzymes in polyamine metabolism,exerting direct influence on polyamine homeostasis regulation.Dysfunctions in these enzymes are intricately linked to inflammatory diseases and cancers.Establishing their three-dimensional structures is essential for exploring enzymatic catalytic mechanisms and designing inhibitors at the atomic level.This article primarily assesses the precision of AlphaFold2 and molecular dynamics simulations in determining the three-dimensional structures of these enzymes,utilizing protein conformation rationality assessment,residue correlation matrix,and other techniques.This provides robust models for subsequent polyamine catabolic metabolism calculations and offers valuable insights for modeling proteins that have yet to acquire crystal structures.展开更多
Microcystinase(MlrA)is a key endopeptidase that catalyzes microcystin degradation without generating harmful byproduct.However,the application of MlrA in the field is primarily impeded by its limited productivity and ...Microcystinase(MlrA)is a key endopeptidase that catalyzes microcystin degradation without generating harmful byproduct.However,the application of MlrA in the field is primarily impeded by its limited productivity and short lifespan.Therefore,the MlrA's function was studied by modelling its structure,which subsequently increased its heterologous expression and high-temperature stability.Results demonstrate that after the irregular sequence at the C-terminus of MlrA was removed,enzyme solubility was significantly decreased.In addition,three fusion tags,namely maltose-binding protein,glutathione S-transferase(GST),and N-utilization substance A(NusA)were used to enhance the overexpression of soluble recombinant MlrA,among which NusA-MlrA exhibited the highest solubility.Moreover,NusA-MlrA was active in pH 4-10 at 20-80℃;even at 80℃,approximately 35.8%of fusion protein remained active.NusA-MlrA retained 89%of MlrA's activity even after 7 d of storage at 50℃;and on day 7,the protein retained>90%of its activity at pH 7.Finally,a stable,soluble,and long-lasting heterologous MlrA was successfully constructed that could eliminate microcystins in Escherichia coli C43(DE3).This study enriched the comprehension of MlrA's structure and enzymatic properties,by particularly addressing the endopeptidase's low expression and short lifespan,which improved its suitability for future applications.展开更多
Dichloromethane(DCM)dehalogenase stands as a crucial enzyme implicated in the degradation of methylene chloride across diverse environmental and biological contexts.However,the unbinding pathways of ligands from DCM d...Dichloromethane(DCM)dehalogenase stands as a crucial enzyme implicated in the degradation of methylene chloride across diverse environmental and biological contexts.However,the unbinding pathways of ligands from DCM dehalogenase remain unexplored.In order to gain a deeper understanding of the binding sites and dissociation pathways of dichloromethane(DCM)and glutathione(GSH)from the DCM dehalogenase,random accelerated molecular dynamics(RAMD)simulations were performed,in which DCM and GSH were forced to leave the active site.The protein structure was predicted using Alphafold2,and the conformations of GSH and DCM in the binding pocket were predicted by docking.A long equilibrium simulation was conducted to validate the structure of the complex.The results show that GSH is most commonly observed in three main pathways,one of which is more important than the other two.In addition,DCM was observed to escape along a unique pathway.The key residues and protein helices of each pathway were identified.The results can provide a theoretical foundation for the subsequent dissociation mechanism of DCM dehalogenase.展开更多
The nuclear pore complex(NPC),one of the largest protein complexes in eukaryotes,serves as a physical gate to regulate nucleocytoplasmic transport.Here,we determined the 8Åresolution cryo-electron microscopic(cry...The nuclear pore complex(NPC),one of the largest protein complexes in eukaryotes,serves as a physical gate to regulate nucleocytoplasmic transport.Here,we determined the 8Åresolution cryo-electron microscopic(cryo-EM)structure of the outer rings containing nuclear ring(NR)and cytoplasmic ring(CR)from the Xenopus laevis NPC,with local resolutions reaching 4.9Å.With the aid of AlphaFold2,we managed to build a pseudoatomic model of the outer rings,including the Y complexes and flanking components.In this most comprehensive and accurate model of outer rings to date,the almost complete Y complex structure exhibits much tighter interaction in the hub region.In addition to two copies of Y complexes,each asymmetric subunit in CR contains five copies of Nup358,two copies of the Nup214 complex,two copies of Nup205 and one copy of newly identified Nup93,while that in NR contains one copy of Nup205,one copy of ELYS and one copy of Nup93.These in-depth structural features represent a great advance in understanding the assembly of NPCs.展开更多
基金National Natural Science Foundation of China(22073023)Natural Science Foundation of Henan Province(242300421134)+1 种基金the Young Backbone Teacher in Colleges and Universities of Henan Province(2021GGJS020)Foundation of State Key Laboratory of Antiviral Drugs。
文摘The acetylpolyamine oxidase(APAO),spermine oxidase(SMO),and spermidine/spermine N1-acetyltransferase(SSAT)are pivotal enzymes in polyamine metabolism,exerting direct influence on polyamine homeostasis regulation.Dysfunctions in these enzymes are intricately linked to inflammatory diseases and cancers.Establishing their three-dimensional structures is essential for exploring enzymatic catalytic mechanisms and designing inhibitors at the atomic level.This article primarily assesses the precision of AlphaFold2 and molecular dynamics simulations in determining the three-dimensional structures of these enzymes,utilizing protein conformation rationality assessment,residue correlation matrix,and other techniques.This provides robust models for subsequent polyamine catabolic metabolism calculations and offers valuable insights for modeling proteins that have yet to acquire crystal structures.
基金Supported by the National Key R&D Program of China(No.2018YFA0903100)the National Natural Science Foundation of China(No.32071601)。
文摘Microcystinase(MlrA)is a key endopeptidase that catalyzes microcystin degradation without generating harmful byproduct.However,the application of MlrA in the field is primarily impeded by its limited productivity and short lifespan.Therefore,the MlrA's function was studied by modelling its structure,which subsequently increased its heterologous expression and high-temperature stability.Results demonstrate that after the irregular sequence at the C-terminus of MlrA was removed,enzyme solubility was significantly decreased.In addition,three fusion tags,namely maltose-binding protein,glutathione S-transferase(GST),and N-utilization substance A(NusA)were used to enhance the overexpression of soluble recombinant MlrA,among which NusA-MlrA exhibited the highest solubility.Moreover,NusA-MlrA was active in pH 4-10 at 20-80℃;even at 80℃,approximately 35.8%of fusion protein remained active.NusA-MlrA retained 89%of MlrA's activity even after 7 d of storage at 50℃;and on day 7,the protein retained>90%of its activity at pH 7.Finally,a stable,soluble,and long-lasting heterologous MlrA was successfully constructed that could eliminate microcystins in Escherichia coli C43(DE3).This study enriched the comprehension of MlrA's structure and enzymatic properties,by particularly addressing the endopeptidase's low expression and short lifespan,which improved its suitability for future applications.
基金National Natural Science Foundation of China(22073030)the Oriental Scholars of Shanghai Universities。
文摘Dichloromethane(DCM)dehalogenase stands as a crucial enzyme implicated in the degradation of methylene chloride across diverse environmental and biological contexts.However,the unbinding pathways of ligands from DCM dehalogenase remain unexplored.In order to gain a deeper understanding of the binding sites and dissociation pathways of dichloromethane(DCM)and glutathione(GSH)from the DCM dehalogenase,random accelerated molecular dynamics(RAMD)simulations were performed,in which DCM and GSH were forced to leave the active site.The protein structure was predicted using Alphafold2,and the conformations of GSH and DCM in the binding pocket were predicted by docking.A long equilibrium simulation was conducted to validate the structure of the complex.The results show that GSH is most commonly observed in three main pathways,one of which is more important than the other two.In addition,DCM was observed to escape along a unique pathway.The key residues and protein helices of each pathway were identified.The results can provide a theoretical foundation for the subsequent dissociation mechanism of DCM dehalogenase.
基金Ministry of Science and Technology of China(2017YFA0504700 to FS and 2016YFA0500201 to CMZ)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB 37040102 to FS),and National Natural Science Foundation of China(31830020 to FS,31520103906 to CMZ)+2 种基金This work was also supported by grants from the National Science Fund for Distinguished Young Scholars(31925026 to FS)National Natural Science Foundation of China(31430051 to CMZ)National Key Research and Development Program of China(2016YFA0100501 to CMZ and 2018YFA0901102 to YZ).
文摘The nuclear pore complex(NPC),one of the largest protein complexes in eukaryotes,serves as a physical gate to regulate nucleocytoplasmic transport.Here,we determined the 8Åresolution cryo-electron microscopic(cryo-EM)structure of the outer rings containing nuclear ring(NR)and cytoplasmic ring(CR)from the Xenopus laevis NPC,with local resolutions reaching 4.9Å.With the aid of AlphaFold2,we managed to build a pseudoatomic model of the outer rings,including the Y complexes and flanking components.In this most comprehensive and accurate model of outer rings to date,the almost complete Y complex structure exhibits much tighter interaction in the hub region.In addition to two copies of Y complexes,each asymmetric subunit in CR contains five copies of Nup358,two copies of the Nup214 complex,two copies of Nup205 and one copy of newly identified Nup93,while that in NR contains one copy of Nup205,one copy of ELYS and one copy of Nup93.These in-depth structural features represent a great advance in understanding the assembly of NPCs.
