Brucella melitensis 7α-hydroxysteroid dehydrogenase(Bm7α-HSDH)catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid.In this work,we investigated the effects of terminal modification(His-tags loca...Brucella melitensis 7α-hydroxysteroid dehydrogenase(Bm7α-HSDH)catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid.In this work,we investigated the effects of terminal modification(His-tags location and terminal truncation)on its catalytic efficiency and thermostability.Compared with C-terminal His-tagged Bm7α-HSDH(C-Bm7α-HSDH),N-Bm7α-HSDH showed a 3.6-fold higher kcat and a 1.3-fold lower Km,resulting in a 7.0-fold higher kcat/Km value toward chenodeoxycholic acid.Circular dichroism spectroscopy indicated that the melting temperature of N-Bm7α-HSDH(46.13℃)was 3.0℃lower than that of C-Bm7α-HSDH(49.13℃).N-Bm7α-HSDH produced 7-oxolithocholic acid in the highest yield of 96.7%in 4 h,whereas the C-Bm7α-HSDH gave 96.4%in 10 h.Moreover,amino acids truncation and His-tag cleave experiments confirmed the C-terminal residues played key roles in catalytic functions.Molecular dynamics simulations further indicated C-terminal His-tagged modification could deform the substrate-binding region to disrupt the enzyme–substrate interactions and catalytic motion.However,the N-terminal His-tag hardly affected the catalytic efficiency due to its location far from the active site of the enzyme.This study provides structural insights into the terminus modifications of hydroxysteroid dehydrogenase on steroid substrate recognition and stabilization,thus affecting its catalytic functions.展开更多
l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesizeβ-hydroxy-α-amino acids with unsatisfactory enzyme activity.Here,we expressed the l-threonine aldolase from Pseudomonas putida KT24...l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesizeβ-hydroxy-α-amino acids with unsatisfactory enzyme activity.Here,we expressed the l-threonine aldolase from Pseudomonas putida KT2440(l-PpTA)in Escherichia coli BL21(DE3)and improved the activity and thermostability by protein engineering.Five amino acid residues(Ser10,His89,Asp93,Arg177,and Arg321)located in the substrate-binding pocket were selected and for mutation.Eight mutants(D93A,D93G,D93M,D93F,D93S,D93Q,D93Y and D93H)with increased enzyme activity were identified and their k_(cat)/K_(M)values showed about 1-7-fold higher than wild-type.Among all the variants,D93H showed the highest catalytic efficiency with 2925 and 4515 s^(−1)mM^(−1)of k_(cat)/K_(M)values toward l-threonine and l-allo-threonine,respectively.In addition,circular dichroism spectrum exhibited that the melting temperature of D93H(54.2℃)was 5℃higher than wild-type(49.2℃).Molecular dynamics simulations illustrated that the D93H variant shortens the distance between the imidazole group of H93 and the hydroxyl group of substrate,which facilitated the proton extraction and promote the enzymatic reaction.This work affords a candidate for the synthesis ofβ-hydroxy-α-amino acids with improved catalytic efficiency and thermostability and provides structural insights into the l-TA family by protein engineering.展开更多
The human Gadd45 protein family plays critical roles in DNA repair,negative growth control,genomic stability,cell cycle checkpoints and apoptosis.Here we report the crystal structure of human Gadd45,revealing a unique...The human Gadd45 protein family plays critical roles in DNA repair,negative growth control,genomic stability,cell cycle checkpoints and apoptosis.Here we report the crystal structure of human Gadd45,revealing a unique dimer formed via a bundle of four parallel helices,involving the most conserved residues among the Gadd45 isoforms.Mutational analysis of human Gadd45 identified a conserved,highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen(PCNA),p21 and cdc2,suggesting that the parallel dimer is the active form for the interaction.Cellular assays indicate that:(1)dimerization of Gadd45 is necessary for apoptosis as well as growth inhibition,and that cell growth inhibition is caused by both cell cycle arrest and apoptosis;(2)a conserved and highly acidic patch on the dimer surface,including the important residues Glu87 and Asp89,is a putative interface for binding proteins related to the cell cycle,DNA repair and apoptosis.These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.展开更多
The natural concentration of bovine lactoferrin C-lobe is low and its separation by proteolytic enzyme digestion is difcult.Here,we expressed the codon-optimized fragment of C-lobe on plasmid pMA0911 with the P_(veg) ...The natural concentration of bovine lactoferrin C-lobe is low and its separation by proteolytic enzyme digestion is difcult.Here,we expressed the codon-optimized fragment of C-lobe on plasmid pMA0911 with the P_(veg) promoter in Bacillus subtilis 168 at 20℃.The yield was 7.5 mg/L,and 90.6%purity was achieved using ammonium sulfate precipitation,Ni–NTA and molecular exclusion.The C-lobe at 10 mg/mL completely inhibited cell growth of Escherichia coli JM109(DE3)and Pseudomonas aeruginosa CGMCC 1.6740,and 48.4%of growth of Staphylococcus aureus CGMCC 1.282,the result is similar to that of 200 ng/mL N-lobe.The minimum inhibitory concentrations of C-lobe were 4,8 and 16 mg/mL,while those of N-lobe were 128,256 and 512μg/mL for E.coli,P.aeruginosa and S.aureus,respectively.This is the frst report on bovine lactoferrin C-lobe expression and the comparative resistance of the recombinant N-and C-lobes in a food-safe strain of B.subtilis.Our fndings ofer the potential to study the structure–function relationship of the N-and C-lobes recombinantly produced in the same host.展开更多
Nascent polypeptide associated complex(NAC)and its two isolated subunits,αNAC and βNAC,play important roles in nascent peptide targeting.We determined a 1.9Åresolution crystal structure of the interaction core ...Nascent polypeptide associated complex(NAC)and its two isolated subunits,αNAC and βNAC,play important roles in nascent peptide targeting.We determined a 1.9Åresolution crystal structure of the interaction core of NAC heterodimer and a 2.4Åresolution crystal structure ofαNAC NAC domain homodimer.These structures provide detailed information of NAC heterodimerization and αNAC homodimerization.We found that the NAC domains of αNAC and βNAC share very similar folding despite of their relative low identity of amino acid sequences.Furthermore,different electric charge distributions of the two subunits at the NAC interface provide an explanation to the observation that the heterodimer of NAC complex is more stable than the single subunit homodimer.In addition,we successfully built a βNAC NAC domain homodimer model based on homologous modeling,suggesting that NAC domain dimerization is a general property of the NAC family.These 3D structures allow further studies on structurefunction relationship of NAC.展开更多
Erratum to:Protein Cell 2011,2(10):814-826 DOI 10.1007/s13238-011-1090-6 Due to typesetting errors,“gadd45”should be“Gadd45γ”in the following places:the article title,the 2^(nd),4^(th) and 5^(th) Gadd45 in the AB...Erratum to:Protein Cell 2011,2(10):814-826 DOI 10.1007/s13238-011-1090-6 Due to typesetting errors,“gadd45”should be“Gadd45γ”in the following places:the article title,the 2^(nd),4^(th) and 5^(th) Gadd45 in the ABSTRACT,the KEYWORDS,the first subti-tle of“RESULTS AND DISCUSSION”section,the legend of Figure 1,the“data collection and processing”of MATERIALS AND METHODS section.展开更多
基金the National Key research and Development Program of China(2018YFA0900302)the National Science Foundation of China(31970045)+2 种基金the National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-12)the Program of Introducing Talents of Discipline to Universities(111-2-06)Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘Brucella melitensis 7α-hydroxysteroid dehydrogenase(Bm7α-HSDH)catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid.In this work,we investigated the effects of terminal modification(His-tags location and terminal truncation)on its catalytic efficiency and thermostability.Compared with C-terminal His-tagged Bm7α-HSDH(C-Bm7α-HSDH),N-Bm7α-HSDH showed a 3.6-fold higher kcat and a 1.3-fold lower Km,resulting in a 7.0-fold higher kcat/Km value toward chenodeoxycholic acid.Circular dichroism spectroscopy indicated that the melting temperature of N-Bm7α-HSDH(46.13℃)was 3.0℃lower than that of C-Bm7α-HSDH(49.13℃).N-Bm7α-HSDH produced 7-oxolithocholic acid in the highest yield of 96.7%in 4 h,whereas the C-Bm7α-HSDH gave 96.4%in 10 h.Moreover,amino acids truncation and His-tag cleave experiments confirmed the C-terminal residues played key roles in catalytic functions.Molecular dynamics simulations further indicated C-terminal His-tagged modification could deform the substrate-binding region to disrupt the enzyme–substrate interactions and catalytic motion.However,the N-terminal His-tag hardly affected the catalytic efficiency due to its location far from the active site of the enzyme.This study provides structural insights into the terminus modifications of hydroxysteroid dehydrogenase on steroid substrate recognition and stabilization,thus affecting its catalytic functions.
基金the National Key research and Development Program of China(2018YFA0900302)the National Science Foundation of China(32271487,31970045)+2 种基金the National Firstclass Discipline Program of Light Industry Technology and Engineering(LITE2018-12)the Program of Introducing Talents of Discipline to Universities(111-2-06)Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesizeβ-hydroxy-α-amino acids with unsatisfactory enzyme activity.Here,we expressed the l-threonine aldolase from Pseudomonas putida KT2440(l-PpTA)in Escherichia coli BL21(DE3)and improved the activity and thermostability by protein engineering.Five amino acid residues(Ser10,His89,Asp93,Arg177,and Arg321)located in the substrate-binding pocket were selected and for mutation.Eight mutants(D93A,D93G,D93M,D93F,D93S,D93Q,D93Y and D93H)with increased enzyme activity were identified and their k_(cat)/K_(M)values showed about 1-7-fold higher than wild-type.Among all the variants,D93H showed the highest catalytic efficiency with 2925 and 4515 s^(−1)mM^(−1)of k_(cat)/K_(M)values toward l-threonine and l-allo-threonine,respectively.In addition,circular dichroism spectrum exhibited that the melting temperature of D93H(54.2℃)was 5℃higher than wild-type(49.2℃).Molecular dynamics simulations illustrated that the D93H variant shortens the distance between the imidazole group of H93 and the hydroxyl group of substrate,which facilitated the proton extraction and promote the enzymatic reaction.This work affords a candidate for the synthesis ofβ-hydroxy-α-amino acids with improved catalytic efficiency and thermostability and provides structural insights into the l-TA family by protein engineering.
基金by the National Basic Research Program(973 Program)(Nos.2006CB806503,2007CB914301 and 2007CB914304)the National Programs for High Technology Research and Development Program(863 Program)(No.2006AA02A322)the National Major Project(Grant No.2009ZX10004-304).
文摘The human Gadd45 protein family plays critical roles in DNA repair,negative growth control,genomic stability,cell cycle checkpoints and apoptosis.Here we report the crystal structure of human Gadd45,revealing a unique dimer formed via a bundle of four parallel helices,involving the most conserved residues among the Gadd45 isoforms.Mutational analysis of human Gadd45 identified a conserved,highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen(PCNA),p21 and cdc2,suggesting that the parallel dimer is the active form for the interaction.Cellular assays indicate that:(1)dimerization of Gadd45 is necessary for apoptosis as well as growth inhibition,and that cell growth inhibition is caused by both cell cycle arrest and apoptosis;(2)a conserved and highly acidic patch on the dimer surface,including the important residues Glu87 and Asp89,is a putative interface for binding proteins related to the cell cycle,DNA repair and apoptosis.These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.
基金This work was supported by the National Key research and Development Program of China(2018YFA0900302)the National Science Foundation of China(31970045)+2 种基金the National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-12)the Program of Introducing Talents of Discipline to Universities(111-2-06)Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘The natural concentration of bovine lactoferrin C-lobe is low and its separation by proteolytic enzyme digestion is difcult.Here,we expressed the codon-optimized fragment of C-lobe on plasmid pMA0911 with the P_(veg) promoter in Bacillus subtilis 168 at 20℃.The yield was 7.5 mg/L,and 90.6%purity was achieved using ammonium sulfate precipitation,Ni–NTA and molecular exclusion.The C-lobe at 10 mg/mL completely inhibited cell growth of Escherichia coli JM109(DE3)and Pseudomonas aeruginosa CGMCC 1.6740,and 48.4%of growth of Staphylococcus aureus CGMCC 1.282,the result is similar to that of 200 ng/mL N-lobe.The minimum inhibitory concentrations of C-lobe were 4,8 and 16 mg/mL,while those of N-lobe were 128,256 and 512μg/mL for E.coli,P.aeruginosa and S.aureus,respectively.This is the frst report on bovine lactoferrin C-lobe expression and the comparative resistance of the recombinant N-and C-lobes in a food-safe strain of B.subtilis.Our fndings ofer the potential to study the structure–function relationship of the N-and C-lobes recombinantly produced in the same host.
基金This work was supported by the National Natural Science Foundation of China(grant No.30730022)the National Basic Research Program(973 Program)(grant Nos.2006CB806503 and 2007CB914304)+1 种基金the National Programs for High Technology Research and Development Program(863 Program)(grant Nos.2006AA02A322 and 2006AA020502)the CAS(China)grant KSCX2-YW-R-05 to Z.R.
文摘Nascent polypeptide associated complex(NAC)and its two isolated subunits,αNAC and βNAC,play important roles in nascent peptide targeting.We determined a 1.9Åresolution crystal structure of the interaction core of NAC heterodimer and a 2.4Åresolution crystal structure ofαNAC NAC domain homodimer.These structures provide detailed information of NAC heterodimerization and αNAC homodimerization.We found that the NAC domains of αNAC and βNAC share very similar folding despite of their relative low identity of amino acid sequences.Furthermore,different electric charge distributions of the two subunits at the NAC interface provide an explanation to the observation that the heterodimer of NAC complex is more stable than the single subunit homodimer.In addition,we successfully built a βNAC NAC domain homodimer model based on homologous modeling,suggesting that NAC domain dimerization is a general property of the NAC family.These 3D structures allow further studies on structurefunction relationship of NAC.
文摘Erratum to:Protein Cell 2011,2(10):814-826 DOI 10.1007/s13238-011-1090-6 Due to typesetting errors,“gadd45”should be“Gadd45γ”in the following places:the article title,the 2^(nd),4^(th) and 5^(th) Gadd45 in the ABSTRACT,the KEYWORDS,the first subti-tle of“RESULTS AND DISCUSSION”section,the legend of Figure 1,the“data collection and processing”of MATERIALS AND METHODS section.
