The Experts below are selected from a list of 75 Experts worldwide ranked by ideXlab platform
Yuzhong Zhang - One of the best experts on this subject based on the ideXlab platform.
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mechanistic insights into elastin degradation by Pseudolysin the major virulence factor of the opportunistic pathogen pseudomonas aeruginosa
Scientific Reports, 2015Co-Authors: Jie Yang, Huilin Zhao, Liyuan Ran, Xiying Zhang, Mei Shi, Baicheng Zhou, Xiulan Chen, Yuzhong ZhangAbstract:Pseudolysin is the most abundant protease secreted by Pseudomonas aeruginosa and is the major extracellular virulence factor of this opportunistic human pathogen. Pseudolysin destroys human tissues by solubilizing elastin. However, the mechanisms by which Pseudolysin binds to and degrades elastin remain elusive. In this study, we investigated the mechanism of action of Pseudolysin on elastin binding and degradation by biochemical assay, microscopy and site-directed mutagenesis. Pseudolysin bound to bovine elastin fibers and preferred to attack peptide bonds with hydrophobic residues at the P1 and P1’ positions in the hydrophobic domains of elastin. The time-course degradation processes of both bovine elastin fibers and cross-linked human tropoelastin by Pseudolysin were further investigated by microscopy. Altogether, the results indicate that elastin degradation by Pseudolysin began with the hydrophobic domains on the fiber surface, followed by the progressive disassembly of macroscopic elastin fibers into primary structural elements. Moreover, our site-directed mutational results indicate that five hydrophobic residues in the S1-S1’ sub-sites played key roles in the binding of Pseudolysin to elastin. This study sheds lights on the pathogenesis of P. aeruginosa infection.
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cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics new insights into relationship between conformational flexibility and
Journal of Biological Chemistry, 2009Co-Authors: Fei Bian, Baicheng Zhou, Xiulan Chen, Hailun He, Yinxin Zeng, Bo Chen, Yuzhong ZhangAbstract:Abstract Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, Pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, kcat/Km (10–40 °C), followed the order Pseudolysin < MCP-02 < E495 with a ratio of ∼1:2:4. MCP-02 and E495 have the same optimal temperature (Topt, 57 °C, 5 °C lower than Pseudolysin) and apparent melting temperature (Tm = 64 °C, ∼10 °C lower than Pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were Pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from Pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.
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cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics new insights into relationship between conformational flexibility and
Journal of Biological Chemistry, 2009Co-Authors: Binbin Xie, Baicheng Zhou, Xiulan Chen, Fei Bian, Yinxin Zeng, Bo Chen, Jun Guo, Xiang Gao, Yuzhong ZhangAbstract:Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, Pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, k(cat)/K(m) (10-40 degrees C), followed the order Pseudolysin < MCP-02 < E495 with a ratio of approximately 1:2:4. MCP-02 and E495 have the same optimal temperature (T(opt), 57 degrees C, 5 degrees C lower than Pseudolysin) and apparent melting temperature (T(m) = 64 degrees C, approximately 10 degrees C lower than Pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were Pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from Pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.
Billy G Hudson - One of the best experts on this subject based on the ideXlab platform.
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glomerular basement membrane identification of a novel disulfide cross linked network of α3 α4 and α5 chains of type iv collagen and its implications for the pathogenesis of alport syndrome
Journal of Biological Chemistry, 1998Co-Authors: Sripad Gunwar, Yoshifumi Ninomiya, Yoshikazu Sado, Milton E Noelken, Fernando Ballester, Billy G HudsonAbstract:Abstract Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated. This was accomplished by using Pseudolysin (EC3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4 °C and the other at 25 °C, and their chain composition was determined by use of monoclonal antibodies. The 4 °C protomers comprise the α1(IV) and α2(IV) chains, whereas the 25 °C protomers comprised mainly α3(IV), α4(IV), and α5(IV) chains along with some α1(IV) and α2(IV) chains. The structure of the 25 °C protomers was examined by electron microscopy and was found to be characterized by a network containing loops and supercoiled triple helices, which are stabilized by disulfide cross-links between α3(IV), α4(IV), and α5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the α3(IV)·α4(IV)·α5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the α5(IV) chain cause defective assembly of not only α5(IV) chain but also the α3(IV) and α4(IV) chains in the GBM of patients with Alport syndrome.
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glomerular basement membrane identification of a novel disulfide cross linked network of α3 α4 and α5 chains of type iv collagen and its implications for the pathogenesis of alport syndrome
Journal of Biological Chemistry, 1998Co-Authors: Sripad Gunwar, Yoshifumi Ninomiya, Yoshikazu Sado, Milton E Noelken, Fernando Ballester, Billy G HudsonAbstract:Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated. This was accomplished by using Pseudolysin (EC 3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4 degrees C and the other at 25 degrees C, and their chain composition was determined by use of monoclonal antibodies. The 4 degrees C protomers comprise the alpha1(IV) and alpha2(IV) chains, whereas the 25 degrees C protomers comprised mainly alpha3(IV), alpha4(IV), and alpha5(IV) chains along with some alpha1(IV) and alpha2(IV) chains. The structure of the 25 degrees C protomers was examined by electron microscopy and was found to be characterized by a network containing loops and supercoiled triple helices, which are stabilized by disulfide cross-links between alpha3(IV), alpha4(IV), and alpha5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the alpha3(IV). alpha4(IV).alpha5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the alpha5(IV) chain cause defective assembly of not only alpha5(IV) chain but also the alpha3(IV) and alpha4(IV) chains in the GBM of patients with Alport syndrome.
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seminiferous tubule basement membrane composition and organization of type iv collagen chains and the linkage of α3 iv and α5 iv chains
Journal of Biological Chemistry, 1997Co-Authors: Tesfamichael Z Kahsai, George C Enders, Sripad Gunwar, Charlott Brunmark, Jorgen Wieslander, Raghuram Kalluri, Milton E Noelken, Jing Zhou, Billy G HudsonAbstract:Abstract Seminiferous tubule basement membrane (STBM) plays an important role in spermatogenesis. In the present study, the composition and structural organization of type IV collagen of bovine STBM was investigated. STBM was found to be composed of all six α-chains of type IV collagen based upon immunocytochemical and biochemical analysis. The content of α3(IV) chain (40%) and the α4(IV) chain (18%) was substantially higher than in any other basement membrane collagen. The supramolecular structure of the six α(IV) chains was investigated using Pseudolysin (EC 3.4.24.26) digestion to excise triple-helical molecules, subsequent collagenase digestion to produce NC1 hexamers and antibody affinity chromatography to resolve populations of NC1 hexamers. The hexamers, which reflect specific arrangements of α(IV) chains, were characterized for their α(IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with α(IV) chain-specific antibodies. Three major hexamer populations were found that represent the classical network of the α1(IV) and α2(IV) chains and two novel networks, one composed of the α1(IV)-α6(IV) chains and the other composed of the α3(IV)-α6(IV) chains. The results establish a structural linkage between the α3(IV) and α5(IV) chains, suggesting a molecular basis for the conundrum in which mutations in the gene encoding the α5(IV) chain cause defective assembly of the α3(IV) chain in the glomerular basement membrane of patients with Alport syndrome.
Sripad Gunwar - One of the best experts on this subject based on the ideXlab platform.
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glomerular basement membrane identification of a novel disulfide cross linked network of α3 α4 and α5 chains of type iv collagen and its implications for the pathogenesis of alport syndrome
Journal of Biological Chemistry, 1998Co-Authors: Sripad Gunwar, Yoshifumi Ninomiya, Yoshikazu Sado, Milton E Noelken, Fernando Ballester, Billy G HudsonAbstract:Abstract Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated. This was accomplished by using Pseudolysin (EC3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4 °C and the other at 25 °C, and their chain composition was determined by use of monoclonal antibodies. The 4 °C protomers comprise the α1(IV) and α2(IV) chains, whereas the 25 °C protomers comprised mainly α3(IV), α4(IV), and α5(IV) chains along with some α1(IV) and α2(IV) chains. The structure of the 25 °C protomers was examined by electron microscopy and was found to be characterized by a network containing loops and supercoiled triple helices, which are stabilized by disulfide cross-links between α3(IV), α4(IV), and α5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the α3(IV)·α4(IV)·α5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the α5(IV) chain cause defective assembly of not only α5(IV) chain but also the α3(IV) and α4(IV) chains in the GBM of patients with Alport syndrome.
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glomerular basement membrane identification of a novel disulfide cross linked network of α3 α4 and α5 chains of type iv collagen and its implications for the pathogenesis of alport syndrome
Journal of Biological Chemistry, 1998Co-Authors: Sripad Gunwar, Yoshifumi Ninomiya, Yoshikazu Sado, Milton E Noelken, Fernando Ballester, Billy G HudsonAbstract:Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated. This was accomplished by using Pseudolysin (EC 3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4 degrees C and the other at 25 degrees C, and their chain composition was determined by use of monoclonal antibodies. The 4 degrees C protomers comprise the alpha1(IV) and alpha2(IV) chains, whereas the 25 degrees C protomers comprised mainly alpha3(IV), alpha4(IV), and alpha5(IV) chains along with some alpha1(IV) and alpha2(IV) chains. The structure of the 25 degrees C protomers was examined by electron microscopy and was found to be characterized by a network containing loops and supercoiled triple helices, which are stabilized by disulfide cross-links between alpha3(IV), alpha4(IV), and alpha5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the alpha3(IV). alpha4(IV).alpha5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the alpha5(IV) chain cause defective assembly of not only alpha5(IV) chain but also the alpha3(IV) and alpha4(IV) chains in the GBM of patients with Alport syndrome.
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seminiferous tubule basement membrane composition and organization of type iv collagen chains and the linkage of α3 iv and α5 iv chains
Journal of Biological Chemistry, 1997Co-Authors: Tesfamichael Z Kahsai, George C Enders, Sripad Gunwar, Charlott Brunmark, Jorgen Wieslander, Raghuram Kalluri, Milton E Noelken, Jing Zhou, Billy G HudsonAbstract:Abstract Seminiferous tubule basement membrane (STBM) plays an important role in spermatogenesis. In the present study, the composition and structural organization of type IV collagen of bovine STBM was investigated. STBM was found to be composed of all six α-chains of type IV collagen based upon immunocytochemical and biochemical analysis. The content of α3(IV) chain (40%) and the α4(IV) chain (18%) was substantially higher than in any other basement membrane collagen. The supramolecular structure of the six α(IV) chains was investigated using Pseudolysin (EC 3.4.24.26) digestion to excise triple-helical molecules, subsequent collagenase digestion to produce NC1 hexamers and antibody affinity chromatography to resolve populations of NC1 hexamers. The hexamers, which reflect specific arrangements of α(IV) chains, were characterized for their α(IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with α(IV) chain-specific antibodies. Three major hexamer populations were found that represent the classical network of the α1(IV) and α2(IV) chains and two novel networks, one composed of the α1(IV)-α6(IV) chains and the other composed of the α3(IV)-α6(IV) chains. The results establish a structural linkage between the α3(IV) and α5(IV) chains, suggesting a molecular basis for the conundrum in which mutations in the gene encoding the α5(IV) chain cause defective assembly of the α3(IV) chain in the glomerular basement membrane of patients with Alport syndrome.
Baicheng Zhou - One of the best experts on this subject based on the ideXlab platform.
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mechanistic insights into elastin degradation by Pseudolysin the major virulence factor of the opportunistic pathogen pseudomonas aeruginosa
Scientific Reports, 2015Co-Authors: Jie Yang, Huilin Zhao, Liyuan Ran, Xiying Zhang, Mei Shi, Baicheng Zhou, Xiulan Chen, Yuzhong ZhangAbstract:Pseudolysin is the most abundant protease secreted by Pseudomonas aeruginosa and is the major extracellular virulence factor of this opportunistic human pathogen. Pseudolysin destroys human tissues by solubilizing elastin. However, the mechanisms by which Pseudolysin binds to and degrades elastin remain elusive. In this study, we investigated the mechanism of action of Pseudolysin on elastin binding and degradation by biochemical assay, microscopy and site-directed mutagenesis. Pseudolysin bound to bovine elastin fibers and preferred to attack peptide bonds with hydrophobic residues at the P1 and P1’ positions in the hydrophobic domains of elastin. The time-course degradation processes of both bovine elastin fibers and cross-linked human tropoelastin by Pseudolysin were further investigated by microscopy. Altogether, the results indicate that elastin degradation by Pseudolysin began with the hydrophobic domains on the fiber surface, followed by the progressive disassembly of macroscopic elastin fibers into primary structural elements. Moreover, our site-directed mutational results indicate that five hydrophobic residues in the S1-S1’ sub-sites played key roles in the binding of Pseudolysin to elastin. This study sheds lights on the pathogenesis of P. aeruginosa infection.
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cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics new insights into relationship between conformational flexibility and
Journal of Biological Chemistry, 2009Co-Authors: Fei Bian, Baicheng Zhou, Xiulan Chen, Hailun He, Yinxin Zeng, Bo Chen, Yuzhong ZhangAbstract:Abstract Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, Pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, kcat/Km (10–40 °C), followed the order Pseudolysin < MCP-02 < E495 with a ratio of ∼1:2:4. MCP-02 and E495 have the same optimal temperature (Topt, 57 °C, 5 °C lower than Pseudolysin) and apparent melting temperature (Tm = 64 °C, ∼10 °C lower than Pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were Pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from Pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.
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cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics new insights into relationship between conformational flexibility and
Journal of Biological Chemistry, 2009Co-Authors: Binbin Xie, Baicheng Zhou, Xiulan Chen, Fei Bian, Yinxin Zeng, Bo Chen, Jun Guo, Xiang Gao, Yuzhong ZhangAbstract:Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, Pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, k(cat)/K(m) (10-40 degrees C), followed the order Pseudolysin < MCP-02 < E495 with a ratio of approximately 1:2:4. MCP-02 and E495 have the same optimal temperature (T(opt), 57 degrees C, 5 degrees C lower than Pseudolysin) and apparent melting temperature (T(m) = 64 degrees C, approximately 10 degrees C lower than Pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were Pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from Pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.
Xiulan Chen - One of the best experts on this subject based on the ideXlab platform.
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mechanistic insights into elastin degradation by Pseudolysin the major virulence factor of the opportunistic pathogen pseudomonas aeruginosa
Scientific Reports, 2015Co-Authors: Jie Yang, Huilin Zhao, Liyuan Ran, Xiying Zhang, Mei Shi, Baicheng Zhou, Xiulan Chen, Yuzhong ZhangAbstract:Pseudolysin is the most abundant protease secreted by Pseudomonas aeruginosa and is the major extracellular virulence factor of this opportunistic human pathogen. Pseudolysin destroys human tissues by solubilizing elastin. However, the mechanisms by which Pseudolysin binds to and degrades elastin remain elusive. In this study, we investigated the mechanism of action of Pseudolysin on elastin binding and degradation by biochemical assay, microscopy and site-directed mutagenesis. Pseudolysin bound to bovine elastin fibers and preferred to attack peptide bonds with hydrophobic residues at the P1 and P1’ positions in the hydrophobic domains of elastin. The time-course degradation processes of both bovine elastin fibers and cross-linked human tropoelastin by Pseudolysin were further investigated by microscopy. Altogether, the results indicate that elastin degradation by Pseudolysin began with the hydrophobic domains on the fiber surface, followed by the progressive disassembly of macroscopic elastin fibers into primary structural elements. Moreover, our site-directed mutational results indicate that five hydrophobic residues in the S1-S1’ sub-sites played key roles in the binding of Pseudolysin to elastin. This study sheds lights on the pathogenesis of P. aeruginosa infection.
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cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics new insights into relationship between conformational flexibility and
Journal of Biological Chemistry, 2009Co-Authors: Fei Bian, Baicheng Zhou, Xiulan Chen, Hailun He, Yinxin Zeng, Bo Chen, Yuzhong ZhangAbstract:Abstract Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, Pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, kcat/Km (10–40 °C), followed the order Pseudolysin < MCP-02 < E495 with a ratio of ∼1:2:4. MCP-02 and E495 have the same optimal temperature (Topt, 57 °C, 5 °C lower than Pseudolysin) and apparent melting temperature (Tm = 64 °C, ∼10 °C lower than Pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were Pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from Pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.
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cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics new insights into relationship between conformational flexibility and
Journal of Biological Chemistry, 2009Co-Authors: Binbin Xie, Baicheng Zhou, Xiulan Chen, Fei Bian, Yinxin Zeng, Bo Chen, Jun Guo, Xiang Gao, Yuzhong ZhangAbstract:Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, Pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, k(cat)/K(m) (10-40 degrees C), followed the order Pseudolysin < MCP-02 < E495 with a ratio of approximately 1:2:4. MCP-02 and E495 have the same optimal temperature (T(opt), 57 degrees C, 5 degrees C lower than Pseudolysin) and apparent melting temperature (T(m) = 64 degrees C, approximately 10 degrees C lower than Pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were Pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from Pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.
