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Shigenori Kanaya - One of the best experts on this subject based on the ideXlab platform.
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hyperthermophilic Subtilisin like proteases from thermococcus kodakarensis
Biotechnology of Microbial Enzymes#R##N#Production Biocatalysis and Industrial Applications, 2017Co-Authors: Ryo Uehara, Shigenori Kanaya, Kazufumi Takano, Yuichi KogaAbstract:Abstract Hyperthermophiles are attractive sources of enzymes owing to their exceptional tolerance to chemical and thermal denaturation. The genome of a hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which optimally grows at 85°C, contains three genes encoding Subtilisin-like serine proteases. We analyzed two of these, Tk-Subtilisin and Tk-SP. Subtilisins from mesophilic bacteria have been widely used in the detergent industry, because of broad substrate specificity and ease of large-scale preparation. Tk-Subtilisin and Tk-SP are approximately 40% identical to these mesophilic bacterial Subtilisins, and exhibit extraordinarily high stability compared with the mesophilic homologs. These two hyperthermophilic Subtilisins are potential candidates for application in biotechnological fields, and will provide good models for the study of maturation and stabilization mechanisms in all Subtilisin-like proteases. Tk-Subtilisin and Tk-SP are synthesized as prepro-enzymes. That is, a signal peptide and a propeptide are attached to the N-terminus of the mature domain, which is then secreted into the external medium as a pro-enzyme, with the assistance of the signal peptide. Most pro-enzymes then mature, undergoing three sequential steps: (1) the folding of mature domain, (2) autoprocessing of propeptide, and (3) degradation of propeptide, as well as bacterial Subtilisin. However, the Tk-Subtilisin and Tk-SP maturation mechanisms are different from those of bacterial Subtilisins with respect to the requirements of the propeptide and Ca2+ ions for folding. Bacterial Subtilisins require a cognate propeptide for folding. In contrast, Tk-Subtilisin requires Ca2+ ions for folding, instead of a propeptide; and Tk-SP requires neither a propeptide, nor Ca2+ ions. Tk-Subtilisin and Tk-SP both require Ca2+ ions for stability, as do bacterial Subtilisins. A crystallographic analysis of Tk-Subtilisin reveals seven Ca2+ ions in its mature domain, while Tk-SP displays two Ca2+ ions in its β-jelly roll domain. Four Ca2+ ions on Tk-Subtilisin’s unique Ca2+ binding loop are responsible for folding, and the other three contribute to stability. The two Ca2+ ions in Tk-SP’s β-jelly roll domain contribute to its hyperstability. Tk-Subtilisin and Tk-SP can degrade persistent proteins, such as abnormal prion proteins (PrPSc), to a level undetectable by western blot analysis, because both are highly tolerant to a wide variety of chemical denaturants, even in the presence of surfactants. Our results suggest that these two hyperstable proteases might be more useful for the medical decontamination of PrPSc than commercially employed enzymes. In this chapter, we summarize the unique maturation and stabilization mechanisms of Tk-Subtilisin and Tk-SP, and discuss their potential for industrial applications.
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Effects of Surfactant and a Hyperthermostable Protease on Infectivity of Scrapie-Infected Mouse Brain Homogenate
Journal of biotechnology & biomaterials, 2015Co-Authors: Azumi Hirata, Shigenori Kanaya, Akikazu Sakudo, Kazufumi Takano, Yuichi KogaAbstract:PrPSc is thought to be the infective agent of TSE, and inactivating the infectivity of PrPSc without using strong reagents is difficult. Although PrPSc is a protease resistant protein, it can be degraded in vitro by the hyperthermophilic protease (Tk-Subtilisin) at temperatures above 65oC through the synergistic effect of heat destabilization of PrP and the high proteolytic activity of the thermostable protease. However, the change in infectivity of the proteasedigested PrPSc is still unknown. Therefore, we used mouse brain homogenate containing PrPSc (SBH) in a bioassay to investigate the loss of infectivity after Tk-Subtilisin digestion. Surprisingly, the Tk-Subtilisin digested SBH retained a high level of infectivity. Despite this, Tk-Subtilisin could still be used for decontamination in highly protein denaturing condition such as in the presence of SDS.
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proteolysis of abnormal prion protein with a thermostable protease from a hyper thermophilic archaeon thermococcus kodakarensis kod1
Biophysical Journal, 2015Co-Authors: Yuichi Koga, Nami Shimizu, Akikazu Sakudo, Shigenori KanayaAbstract:The abnormal prion protein (PrPSc: scrapie-associated prion protein) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrPSc is highly resistant to normal sterilization procedures, the decontamination of PrPSc is a significant public health issue. Tk-Subtilisin is a Subtilisin-like serine protease identified from a hyperthermophilic archaeon Thermococcus kodakarensis KOD1. Among the Subtilisin family of proteases, Tk-Subtilisin has significant high heat stability with its highest specific activity at 90°C and a half-life of 50 min at 100°C. In the present study, a hyper-thermostable protease, Tk-Subtilisin, was used to degrade PrPSc. Although PrPSc is known to be resistant toward proteolytic enzymes, Tk-Subtilisin was able to degrade PrPSc under extreme conditions. The level of PrPSc in brain homogenates was found to decrease significantly in vitro following Tk-Subtilisin treatment at 100°C, whereas some protease resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-Subtilisin were required to degrade PrPSc at 100°C and pH 8.0. In addition, Tk-Subtilisin was observed to degrade PrPSc in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrPSc have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrPSc degradation.
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increase in activation rate of pro tk Subtilisin by a single nonpolar to polar amino acid substitution at the hydrophobic core of the propeptide domain
Protein Science, 2013Co-Authors: Kota Yuzaki, Ryo Uehara, Yuichi Koga, Yudai Sanda, Dong-ju You, Shigenori KanayaAbstract:Tk-Subtilisin (Gly70-Gly398) is a Subtilisin homolog from Thermococcus kodakarensis. Active Tk-Subtilisin is produced from its inactive precursor, Pro-Tk-Subtilisin (Gly1-Gly398), by autoprocessing and degradation of the propeptide (Tk-propeptide, Gly1-Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk-propeptide. Tk-propeptide is stabilized by the hydrophobic core. To examine whether a single nonpolar-to-polar amino acid substitution at this core affects the activation rate of Pro-Tk-Subtilisin, the Pro-Tk-Subtilisin derivative with the Phe17→His mutation (Pro-F17H), Tk-propeptide derivative with the same mutation (F17H-propeptide), and two active-site mutants of Pro-F17H (Pro-F17H/S324A and Pro-F17H/S324C) were constructed. The crystal structure of Pro-F17H/S324A was nearly identical to that of Pro-S324A, indicating that the mutation does not affect the structure of Pro-Tk-Subtilisin. The refolding rate of Pro-F17H/S324A and autoprocessing rate of Pro-F17H/S324C were also nearly identical to those of their parent proteins (Pro-S324A and Pro-S324C). However, the activation rate of Pro-F17H greatly increased when compared with that of Pro-Tk-Subtilisin, such that Pro-F17H is efficiently activated even at 40°C. The far-UV circular dichroism spectrum of F17H-propeptide did not exhibit a broad trough at 205–230 nm, which is observed in the spectrum of Tk-propeptide. F17H-propeptide is more susceptible to chymotryptic degradation than Tk-propeptide. These results suggest that F17H-propeptide is unfolded in an isolated form and is therefore rapidly degraded by Tk-Subtilisin. Thus, destabilization of the hydrophobic core of Tk-propeptide by a nonpolar-to-polar amino acid substitution is an effective way to increase the activation rate of Pro-Tk-Subtilisin.
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requirement of ca 2 ions for the hyperthermostability of tk Subtilisin from thermococcus kodakarensis
Biochemistry, 2012Co-Authors: Ryo Uehara, Shunichi Tanaka, Yuichi Koga, Yuki Takeuchi, Kazufumi Takano, Shigenori KanayaAbstract:Tk-Subtilisin, a hyperthermostable Subtilisin-like serine protease from Thermococcus kodakarensis, matures from the inactive precursor, Pro-Tk-Subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro). It contains seven Ca2+ ions. Four of them (Ca2–Ca5) are responsible for folding of Tk-Subtilisin. In this study, to clarify the role of the other three Ca2+ ions (Ca1, Ca6, and Ca7), we constructed Pro-TKS derivatives lacking the Ca1 ion (Pro-TKS/ΔCa1), Ca6 ion (Pro-TKS/ΔCa6), and Ca7 ion (Pro-TKS/ΔCa7), and their active site mutants (Pro-S324A/ΔCa1, Pro-S324A/ΔCa6, and Pro-S324A/ΔCa7, respectively). Pro-TKS/ΔCa6 and Pro-TKS/ΔCa7 fully matured into their active forms upon incubation at 80 °C for 30 min as did Pro-TKS. The mature enzymes were as active as Tk-Subtilisin at 80 °C, indicating that the Ca6 and Ca7 ions are not important for activity. In contrast, Pro-TKS/ΔCa1 matured poorly at 80 °C because of the instability of its mature domain. The enzymatic activity of Tk-Subtilisin...
Yuichi Koga - One of the best experts on this subject based on the ideXlab platform.
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hyperthermophilic Subtilisin like proteases from thermococcus kodakarensis
Biotechnology of Microbial Enzymes#R##N#Production Biocatalysis and Industrial Applications, 2017Co-Authors: Ryo Uehara, Shigenori Kanaya, Kazufumi Takano, Yuichi KogaAbstract:Abstract Hyperthermophiles are attractive sources of enzymes owing to their exceptional tolerance to chemical and thermal denaturation. The genome of a hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which optimally grows at 85°C, contains three genes encoding Subtilisin-like serine proteases. We analyzed two of these, Tk-Subtilisin and Tk-SP. Subtilisins from mesophilic bacteria have been widely used in the detergent industry, because of broad substrate specificity and ease of large-scale preparation. Tk-Subtilisin and Tk-SP are approximately 40% identical to these mesophilic bacterial Subtilisins, and exhibit extraordinarily high stability compared with the mesophilic homologs. These two hyperthermophilic Subtilisins are potential candidates for application in biotechnological fields, and will provide good models for the study of maturation and stabilization mechanisms in all Subtilisin-like proteases. Tk-Subtilisin and Tk-SP are synthesized as prepro-enzymes. That is, a signal peptide and a propeptide are attached to the N-terminus of the mature domain, which is then secreted into the external medium as a pro-enzyme, with the assistance of the signal peptide. Most pro-enzymes then mature, undergoing three sequential steps: (1) the folding of mature domain, (2) autoprocessing of propeptide, and (3) degradation of propeptide, as well as bacterial Subtilisin. However, the Tk-Subtilisin and Tk-SP maturation mechanisms are different from those of bacterial Subtilisins with respect to the requirements of the propeptide and Ca2+ ions for folding. Bacterial Subtilisins require a cognate propeptide for folding. In contrast, Tk-Subtilisin requires Ca2+ ions for folding, instead of a propeptide; and Tk-SP requires neither a propeptide, nor Ca2+ ions. Tk-Subtilisin and Tk-SP both require Ca2+ ions for stability, as do bacterial Subtilisins. A crystallographic analysis of Tk-Subtilisin reveals seven Ca2+ ions in its mature domain, while Tk-SP displays two Ca2+ ions in its β-jelly roll domain. Four Ca2+ ions on Tk-Subtilisin’s unique Ca2+ binding loop are responsible for folding, and the other three contribute to stability. The two Ca2+ ions in Tk-SP’s β-jelly roll domain contribute to its hyperstability. Tk-Subtilisin and Tk-SP can degrade persistent proteins, such as abnormal prion proteins (PrPSc), to a level undetectable by western blot analysis, because both are highly tolerant to a wide variety of chemical denaturants, even in the presence of surfactants. Our results suggest that these two hyperstable proteases might be more useful for the medical decontamination of PrPSc than commercially employed enzymes. In this chapter, we summarize the unique maturation and stabilization mechanisms of Tk-Subtilisin and Tk-SP, and discuss their potential for industrial applications.
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Effects of Surfactant and a Hyperthermostable Protease on Infectivity of Scrapie-Infected Mouse Brain Homogenate
Journal of biotechnology & biomaterials, 2015Co-Authors: Azumi Hirata, Shigenori Kanaya, Akikazu Sakudo, Kazufumi Takano, Yuichi KogaAbstract:PrPSc is thought to be the infective agent of TSE, and inactivating the infectivity of PrPSc without using strong reagents is difficult. Although PrPSc is a protease resistant protein, it can be degraded in vitro by the hyperthermophilic protease (Tk-Subtilisin) at temperatures above 65oC through the synergistic effect of heat destabilization of PrP and the high proteolytic activity of the thermostable protease. However, the change in infectivity of the proteasedigested PrPSc is still unknown. Therefore, we used mouse brain homogenate containing PrPSc (SBH) in a bioassay to investigate the loss of infectivity after Tk-Subtilisin digestion. Surprisingly, the Tk-Subtilisin digested SBH retained a high level of infectivity. Despite this, Tk-Subtilisin could still be used for decontamination in highly protein denaturing condition such as in the presence of SDS.
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proteolysis of abnormal prion protein with a thermostable protease from a hyper thermophilic archaeon thermococcus kodakarensis kod1
Biophysical Journal, 2015Co-Authors: Yuichi Koga, Nami Shimizu, Akikazu Sakudo, Shigenori KanayaAbstract:The abnormal prion protein (PrPSc: scrapie-associated prion protein) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrPSc is highly resistant to normal sterilization procedures, the decontamination of PrPSc is a significant public health issue. Tk-Subtilisin is a Subtilisin-like serine protease identified from a hyperthermophilic archaeon Thermococcus kodakarensis KOD1. Among the Subtilisin family of proteases, Tk-Subtilisin has significant high heat stability with its highest specific activity at 90°C and a half-life of 50 min at 100°C. In the present study, a hyper-thermostable protease, Tk-Subtilisin, was used to degrade PrPSc. Although PrPSc is known to be resistant toward proteolytic enzymes, Tk-Subtilisin was able to degrade PrPSc under extreme conditions. The level of PrPSc in brain homogenates was found to decrease significantly in vitro following Tk-Subtilisin treatment at 100°C, whereas some protease resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-Subtilisin were required to degrade PrPSc at 100°C and pH 8.0. In addition, Tk-Subtilisin was observed to degrade PrPSc in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrPSc have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrPSc degradation.
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increase in activation rate of pro tk Subtilisin by a single nonpolar to polar amino acid substitution at the hydrophobic core of the propeptide domain
Protein Science, 2013Co-Authors: Kota Yuzaki, Ryo Uehara, Yuichi Koga, Yudai Sanda, Dong-ju You, Shigenori KanayaAbstract:Tk-Subtilisin (Gly70-Gly398) is a Subtilisin homolog from Thermococcus kodakarensis. Active Tk-Subtilisin is produced from its inactive precursor, Pro-Tk-Subtilisin (Gly1-Gly398), by autoprocessing and degradation of the propeptide (Tk-propeptide, Gly1-Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk-propeptide. Tk-propeptide is stabilized by the hydrophobic core. To examine whether a single nonpolar-to-polar amino acid substitution at this core affects the activation rate of Pro-Tk-Subtilisin, the Pro-Tk-Subtilisin derivative with the Phe17→His mutation (Pro-F17H), Tk-propeptide derivative with the same mutation (F17H-propeptide), and two active-site mutants of Pro-F17H (Pro-F17H/S324A and Pro-F17H/S324C) were constructed. The crystal structure of Pro-F17H/S324A was nearly identical to that of Pro-S324A, indicating that the mutation does not affect the structure of Pro-Tk-Subtilisin. The refolding rate of Pro-F17H/S324A and autoprocessing rate of Pro-F17H/S324C were also nearly identical to those of their parent proteins (Pro-S324A and Pro-S324C). However, the activation rate of Pro-F17H greatly increased when compared with that of Pro-Tk-Subtilisin, such that Pro-F17H is efficiently activated even at 40°C. The far-UV circular dichroism spectrum of F17H-propeptide did not exhibit a broad trough at 205–230 nm, which is observed in the spectrum of Tk-propeptide. F17H-propeptide is more susceptible to chymotryptic degradation than Tk-propeptide. These results suggest that F17H-propeptide is unfolded in an isolated form and is therefore rapidly degraded by Tk-Subtilisin. Thus, destabilization of the hydrophobic core of Tk-propeptide by a nonpolar-to-polar amino acid substitution is an effective way to increase the activation rate of Pro-Tk-Subtilisin.
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requirement of ca 2 ions for the hyperthermostability of tk Subtilisin from thermococcus kodakarensis
Biochemistry, 2012Co-Authors: Ryo Uehara, Shunichi Tanaka, Yuichi Koga, Yuki Takeuchi, Kazufumi Takano, Shigenori KanayaAbstract:Tk-Subtilisin, a hyperthermostable Subtilisin-like serine protease from Thermococcus kodakarensis, matures from the inactive precursor, Pro-Tk-Subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro). It contains seven Ca2+ ions. Four of them (Ca2–Ca5) are responsible for folding of Tk-Subtilisin. In this study, to clarify the role of the other three Ca2+ ions (Ca1, Ca6, and Ca7), we constructed Pro-TKS derivatives lacking the Ca1 ion (Pro-TKS/ΔCa1), Ca6 ion (Pro-TKS/ΔCa6), and Ca7 ion (Pro-TKS/ΔCa7), and their active site mutants (Pro-S324A/ΔCa1, Pro-S324A/ΔCa6, and Pro-S324A/ΔCa7, respectively). Pro-TKS/ΔCa6 and Pro-TKS/ΔCa7 fully matured into their active forms upon incubation at 80 °C for 30 min as did Pro-TKS. The mature enzymes were as active as Tk-Subtilisin at 80 °C, indicating that the Ca6 and Ca7 ions are not important for activity. In contrast, Pro-TKS/ΔCa1 matured poorly at 80 °C because of the instability of its mature domain. The enzymatic activity of Tk-Subtilisin...
Kazufumi Takano - One of the best experts on this subject based on the ideXlab platform.
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hyperthermophilic Subtilisin like proteases from thermococcus kodakarensis
Biotechnology of Microbial Enzymes#R##N#Production Biocatalysis and Industrial Applications, 2017Co-Authors: Ryo Uehara, Shigenori Kanaya, Kazufumi Takano, Yuichi KogaAbstract:Abstract Hyperthermophiles are attractive sources of enzymes owing to their exceptional tolerance to chemical and thermal denaturation. The genome of a hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which optimally grows at 85°C, contains three genes encoding Subtilisin-like serine proteases. We analyzed two of these, Tk-Subtilisin and Tk-SP. Subtilisins from mesophilic bacteria have been widely used in the detergent industry, because of broad substrate specificity and ease of large-scale preparation. Tk-Subtilisin and Tk-SP are approximately 40% identical to these mesophilic bacterial Subtilisins, and exhibit extraordinarily high stability compared with the mesophilic homologs. These two hyperthermophilic Subtilisins are potential candidates for application in biotechnological fields, and will provide good models for the study of maturation and stabilization mechanisms in all Subtilisin-like proteases. Tk-Subtilisin and Tk-SP are synthesized as prepro-enzymes. That is, a signal peptide and a propeptide are attached to the N-terminus of the mature domain, which is then secreted into the external medium as a pro-enzyme, with the assistance of the signal peptide. Most pro-enzymes then mature, undergoing three sequential steps: (1) the folding of mature domain, (2) autoprocessing of propeptide, and (3) degradation of propeptide, as well as bacterial Subtilisin. However, the Tk-Subtilisin and Tk-SP maturation mechanisms are different from those of bacterial Subtilisins with respect to the requirements of the propeptide and Ca2+ ions for folding. Bacterial Subtilisins require a cognate propeptide for folding. In contrast, Tk-Subtilisin requires Ca2+ ions for folding, instead of a propeptide; and Tk-SP requires neither a propeptide, nor Ca2+ ions. Tk-Subtilisin and Tk-SP both require Ca2+ ions for stability, as do bacterial Subtilisins. A crystallographic analysis of Tk-Subtilisin reveals seven Ca2+ ions in its mature domain, while Tk-SP displays two Ca2+ ions in its β-jelly roll domain. Four Ca2+ ions on Tk-Subtilisin’s unique Ca2+ binding loop are responsible for folding, and the other three contribute to stability. The two Ca2+ ions in Tk-SP’s β-jelly roll domain contribute to its hyperstability. Tk-Subtilisin and Tk-SP can degrade persistent proteins, such as abnormal prion proteins (PrPSc), to a level undetectable by western blot analysis, because both are highly tolerant to a wide variety of chemical denaturants, even in the presence of surfactants. Our results suggest that these two hyperstable proteases might be more useful for the medical decontamination of PrPSc than commercially employed enzymes. In this chapter, we summarize the unique maturation and stabilization mechanisms of Tk-Subtilisin and Tk-SP, and discuss their potential for industrial applications.
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Effects of Surfactant and a Hyperthermostable Protease on Infectivity of Scrapie-Infected Mouse Brain Homogenate
Journal of biotechnology & biomaterials, 2015Co-Authors: Azumi Hirata, Shigenori Kanaya, Akikazu Sakudo, Kazufumi Takano, Yuichi KogaAbstract:PrPSc is thought to be the infective agent of TSE, and inactivating the infectivity of PrPSc without using strong reagents is difficult. Although PrPSc is a protease resistant protein, it can be degraded in vitro by the hyperthermophilic protease (Tk-Subtilisin) at temperatures above 65oC through the synergistic effect of heat destabilization of PrP and the high proteolytic activity of the thermostable protease. However, the change in infectivity of the proteasedigested PrPSc is still unknown. Therefore, we used mouse brain homogenate containing PrPSc (SBH) in a bioassay to investigate the loss of infectivity after Tk-Subtilisin digestion. Surprisingly, the Tk-Subtilisin digested SBH retained a high level of infectivity. Despite this, Tk-Subtilisin could still be used for decontamination in highly protein denaturing condition such as in the presence of SDS.
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requirement of ca 2 ions for the hyperthermostability of tk Subtilisin from thermococcus kodakarensis
Biochemistry, 2012Co-Authors: Ryo Uehara, Shunichi Tanaka, Yuichi Koga, Yuki Takeuchi, Kazufumi Takano, Shigenori KanayaAbstract:Tk-Subtilisin, a hyperthermostable Subtilisin-like serine protease from Thermococcus kodakarensis, matures from the inactive precursor, Pro-Tk-Subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro). It contains seven Ca2+ ions. Four of them (Ca2–Ca5) are responsible for folding of Tk-Subtilisin. In this study, to clarify the role of the other three Ca2+ ions (Ca1, Ca6, and Ca7), we constructed Pro-TKS derivatives lacking the Ca1 ion (Pro-TKS/ΔCa1), Ca6 ion (Pro-TKS/ΔCa6), and Ca7 ion (Pro-TKS/ΔCa7), and their active site mutants (Pro-S324A/ΔCa1, Pro-S324A/ΔCa6, and Pro-S324A/ΔCa7, respectively). Pro-TKS/ΔCa6 and Pro-TKS/ΔCa7 fully matured into their active forms upon incubation at 80 °C for 30 min as did Pro-TKS. The mature enzymes were as active as Tk-Subtilisin at 80 °C, indicating that the Ca6 and Ca7 ions are not important for activity. In contrast, Pro-TKS/ΔCa1 matured poorly at 80 °C because of the instability of its mature domain. The enzymatic activity of Tk-Subtilisin...
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Subtilisin like serine protease from hyperthermophilic archaeon thermococcus kodakaraensis with n and c terminal propeptides
Protein Engineering Design & Selection, 2010Co-Authors: Tita Foophow, Shunichi Tanaka, Yuichi Koga, Kazufumi Takano, Shigenori KanayaAbstract:: The genome of the hyperthermophilic archaeon Thermococcus kodakaraensis contains three genes encoding Subtilisin-like serine proteases, Tk-1689, Tk-0076 and Tk-Subtilisin. Of them, the structure and function of Tk-Subtilisin have been extensively studied. To examine whether Tk-1689 is matured to an active form and functions as a hyperthermostable protease as is Tk-Subtilisin, the gene encoding the Tk-1689 derivative without a putative N-terminal signal sequence, termed Pro-Tk-SP, was overexpressed in Escherichia coli. Pro-Tk-SP is composed of 640 amino acid residues and its molecular mass is 68.6 kDa. The recombinant protein was purified, however, as an active 44 kDa protease, termed Tk-SP, which lacks the N-terminal 113 and C-terminal 101 amino acid residues. This result suggests that Pro-Tk-SP consists of an N-terminal propeptide (Ala1-Ala113), a mature domain (Tk-SP, Val114-Val539) and a C-terminal propeptide (Asp540-Gly640). Like Tk-Subtilisin, Tk-SP showed a broad substrate specificity and was highly thermostable. Its optimum temperature for activity was approximately 100 degrees C and its half-life at 100 degrees C was 100 min. It was fully resistant to treatment with 5% SDS, 8 M urea or 10% Triton X-100. However, unlike Tk-Subtilisin and bacterial Subtilisins, Tk-SP requires neither Ca2+ nor propeptide for folding. As a result, Tk-SP was fully active even in the presence of 10 mM EDTA. Thus, Tk-SP has a great advantage over other proteases in high resistance to heat, denaturants, detergents and chelating agents and therefore has great potential for application in biotechnology fields.
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requirement of left handed glycine residue for high stability of the tk Subtilisin propeptide as revealed by mutational and crystallographic analyses
Journal of Molecular Biology, 2007Co-Authors: M A Pulido, Shunichi Tanaka, Yuichi Koga, Kazufumi Takano, Dong-ju You, Chutima Sringiew, Hiroyoshi Matsumura, Shigenori KanayaAbstract:Abstract Tk-Subtilisin [the mature domain of Pro-Tk-Subtilisin in active form (Gly70-Gly398)] from the hyperthermophilic archaeon Thermococcus kodakaraensis is matured from Pro-Tk-Subtilisin [a Subtilisin homologue from T. kodakaraensis in pro form (Gly1-Gly398)] upon autoprocessing and degradation of propeptide. Pro-Tk-Subtilisin is characterized by extremely slow maturation at mild temperatures, but this maturation rate is greatly increased by a single Gly56 → Ser mutation in the propeptide region. To analyze the role of Gly56, which assumes a left-handed conformation, Pro-Tk-Subtilisin variants with complete amino acid substitutions at Gly56 were constructed. A comparison of their halo-forming activities suggests that all variants, except for Pro-G56W [Pro-G56X, Pro-Tk-Subtilisin with Gly56 → X mutation (X = any amino acid)], mature faster than WT. Pro-G56W and Pro-G56E with the lowest and highest maturation rates, respectively, among 19 variants, as well as WT and Pro-G56S, were overproduced, purified, and characterized. SDS-PAGE analyses and Tk-Subtilisin activity assay indicated that their maturation rates increased in the order WT ≤ Pro-G56W G56S-propeptide > G56E-propeptide, indicating that they are inversely correlated with the maturation rates of Pro7-Tk-Subtilisin and its derivatives. The crystal structures of these propeptides determined in complex with S324A-Subtilisin indicate that the conformation of the propeptide is altered by the mutation, such that nonglycine residues at position 56 assume a right-handed conformation and hydrophobic interactions at the core region decrease. These results indicate that Gly56 is required in stabilizing the propeptide fold. Stabilization of this fold leads to strong binding of Tk-propeptide to Tk-Subtilisin, high resistance of Tk-propeptide to proteolytic degradation, and slow maturation of Pro-Tk-Subtilisin.
Riad Lutfi - One of the best experts on this subject based on the ideXlab platform.
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proprotein convertase Subtilisin kexin type 9 loss of function is detrimental to the juvenile host with septic shock
Critical Care Medicine, 2020Co-Authors: Mihir R Atreya, Brynne Whitacre, Natalie Z Cvijanovich, Michael T Bigham, Neal J Thomas, Adam Schwarz, Scott L Weiss, Julie C Fitzgerald, Geoffrey L Allen, Riad LutfiAbstract:Objectives Proprotein convertase Subtilisin/kexin type 9 is a central regulator of lipid metabolism and has been implicated in regulating the host response to sepsis. Proprotein convertase Subtilisin/kexin type 9 loss-of-function is associated with improved sepsis outcomes in the adult host through increased hepatic bacterial clearance. Thus, there is interest in leveraging proprotein convertase Subtilisin/kexin type 9 inhibitors as a therapeutic strategy in adults with sepsis. We sought to validate this association in children with septic shock and in a juvenile murine model of sepsis. Design Prospectively enrolled cohort of children with septic shock; experimental mice. Setting Seventeen participating institutions; research laboratory. Patients and subjects Five-hundred twenty-two children with septic shock; juvenile (14 d old) and adult (10-14 wk) mice with constitutive proprotein convertase Subtilisin/kexin type 9 null and wildtype control mice (C57BL/6). Interventions Proprotein convertase Subtilisin/kexin type 9 single-nucleotide polymorphisms, serum proprotein convertase Subtilisin/kexin type 9, and lipid profiles in patients. Cecal slurry murine model of sepsis; survival studies in juvenile and adult mice, assessment of lipoprotein fractions, bacterial burden, and inflammation in juvenile mice. Measurements and main results PCSK9 loss-of-function genetic variants were independently associated with increased odds of complicated course and mortality in children with septic shock. PCSK9, low-density lipoprotein, and high-density lipoprotein concentrations were lower among patients with complicated course relative to those without. PCSK9concentrations negatively correlated with proinflammatory cytokine interleukin-8. Proprotein convertase Subtilisin/kexin type 9 loss-of-function decreased survival in juvenile mice, but increased survival in adult mice with sepsis. PCSK9 loss-of-function resulted in low lipoproteins and decreased hepatic bacterial burden in juvenile mice. Conclusions In contrast to the adult host, proprotein convertase Subtilisin/kexin type 9 loss-of-function is detrimental to the juvenile host with septic shock. PCSK9 loss-of-function, in the context of low lipoproteins, may result in reduced hepatic bacterial clearance in the juvenile host with septic shock. Our data indicate that children should be excluded in sepsis clinical trials involving proprotein convertase Subtilisin/kexin type 9 inhibitors.
Shunichi Tanaka - One of the best experts on this subject based on the ideXlab platform.
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requirement of ca 2 ions for the hyperthermostability of tk Subtilisin from thermococcus kodakarensis
Biochemistry, 2012Co-Authors: Ryo Uehara, Shunichi Tanaka, Yuichi Koga, Yuki Takeuchi, Kazufumi Takano, Shigenori KanayaAbstract:Tk-Subtilisin, a hyperthermostable Subtilisin-like serine protease from Thermococcus kodakarensis, matures from the inactive precursor, Pro-Tk-Subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro). It contains seven Ca2+ ions. Four of them (Ca2–Ca5) are responsible for folding of Tk-Subtilisin. In this study, to clarify the role of the other three Ca2+ ions (Ca1, Ca6, and Ca7), we constructed Pro-TKS derivatives lacking the Ca1 ion (Pro-TKS/ΔCa1), Ca6 ion (Pro-TKS/ΔCa6), and Ca7 ion (Pro-TKS/ΔCa7), and their active site mutants (Pro-S324A/ΔCa1, Pro-S324A/ΔCa6, and Pro-S324A/ΔCa7, respectively). Pro-TKS/ΔCa6 and Pro-TKS/ΔCa7 fully matured into their active forms upon incubation at 80 °C for 30 min as did Pro-TKS. The mature enzymes were as active as Tk-Subtilisin at 80 °C, indicating that the Ca6 and Ca7 ions are not important for activity. In contrast, Pro-TKS/ΔCa1 matured poorly at 80 °C because of the instability of its mature domain. The enzymatic activity of Tk-Subtilisin...
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Subtilisin like serine protease from hyperthermophilic archaeon thermococcus kodakaraensis with n and c terminal propeptides
Protein Engineering Design & Selection, 2010Co-Authors: Tita Foophow, Shunichi Tanaka, Yuichi Koga, Kazufumi Takano, Shigenori KanayaAbstract:: The genome of the hyperthermophilic archaeon Thermococcus kodakaraensis contains three genes encoding Subtilisin-like serine proteases, Tk-1689, Tk-0076 and Tk-Subtilisin. Of them, the structure and function of Tk-Subtilisin have been extensively studied. To examine whether Tk-1689 is matured to an active form and functions as a hyperthermostable protease as is Tk-Subtilisin, the gene encoding the Tk-1689 derivative without a putative N-terminal signal sequence, termed Pro-Tk-SP, was overexpressed in Escherichia coli. Pro-Tk-SP is composed of 640 amino acid residues and its molecular mass is 68.6 kDa. The recombinant protein was purified, however, as an active 44 kDa protease, termed Tk-SP, which lacks the N-terminal 113 and C-terminal 101 amino acid residues. This result suggests that Pro-Tk-SP consists of an N-terminal propeptide (Ala1-Ala113), a mature domain (Tk-SP, Val114-Val539) and a C-terminal propeptide (Asp540-Gly640). Like Tk-Subtilisin, Tk-SP showed a broad substrate specificity and was highly thermostable. Its optimum temperature for activity was approximately 100 degrees C and its half-life at 100 degrees C was 100 min. It was fully resistant to treatment with 5% SDS, 8 M urea or 10% Triton X-100. However, unlike Tk-Subtilisin and bacterial Subtilisins, Tk-SP requires neither Ca2+ nor propeptide for folding. As a result, Tk-SP was fully active even in the presence of 10 mM EDTA. Thus, Tk-SP has a great advantage over other proteases in high resistance to heat, denaturants, detergents and chelating agents and therefore has great potential for application in biotechnology fields.
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requirement of left handed glycine residue for high stability of the tk Subtilisin propeptide as revealed by mutational and crystallographic analyses
Journal of Molecular Biology, 2007Co-Authors: M A Pulido, Shunichi Tanaka, Yuichi Koga, Kazufumi Takano, Dong-ju You, Chutima Sringiew, Hiroyoshi Matsumura, Shigenori KanayaAbstract:Abstract Tk-Subtilisin [the mature domain of Pro-Tk-Subtilisin in active form (Gly70-Gly398)] from the hyperthermophilic archaeon Thermococcus kodakaraensis is matured from Pro-Tk-Subtilisin [a Subtilisin homologue from T. kodakaraensis in pro form (Gly1-Gly398)] upon autoprocessing and degradation of propeptide. Pro-Tk-Subtilisin is characterized by extremely slow maturation at mild temperatures, but this maturation rate is greatly increased by a single Gly56 → Ser mutation in the propeptide region. To analyze the role of Gly56, which assumes a left-handed conformation, Pro-Tk-Subtilisin variants with complete amino acid substitutions at Gly56 were constructed. A comparison of their halo-forming activities suggests that all variants, except for Pro-G56W [Pro-G56X, Pro-Tk-Subtilisin with Gly56 → X mutation (X = any amino acid)], mature faster than WT. Pro-G56W and Pro-G56E with the lowest and highest maturation rates, respectively, among 19 variants, as well as WT and Pro-G56S, were overproduced, purified, and characterized. SDS-PAGE analyses and Tk-Subtilisin activity assay indicated that their maturation rates increased in the order WT ≤ Pro-G56W G56S-propeptide > G56E-propeptide, indicating that they are inversely correlated with the maturation rates of Pro7-Tk-Subtilisin and its derivatives. The crystal structures of these propeptides determined in complex with S324A-Subtilisin indicate that the conformation of the propeptide is altered by the mutation, such that nonglycine residues at position 56 assume a right-handed conformation and hydrophobic interactions at the core region decrease. These results indicate that Gly56 is required in stabilizing the propeptide fold. Stabilization of this fold leads to strong binding of Tk-propeptide to Tk-Subtilisin, high resistance of Tk-propeptide to proteolytic degradation, and slow maturation of Pro-Tk-Subtilisin.
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ca2 dependent maturation of Subtilisin from a hyperthermophilic archaeon thermococcus kodakaraensis the propeptide is a potent inhibitor of the mature domain but is not required for its folding
Applied and Environmental Microbiology, 2006Co-Authors: M A Pulido, Shunichi Tanaka, Yuichi Koga, Kazufumi Takano, Kenji Saito, Masaaki Morikawa, Shigenori KanayaAbstract:Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the Subtilisin family. T. kodakaraensis Subtilisin in a proform (T. kodakaraensis pro-Subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-Subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-Subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-Subtilisin*) but forms an inactive complex with T. kodakaraensis mat-Subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-Subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-Subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-Subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-Subtilisin*, indicating that T. kodakaraensis mat-Subtilisin does not require T. kodakaraensis propeptide for folding. However, only ∼5% of T. kodakaraensis mat-Subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-Subtilisin* and noncompetitively inhibited its activity with a Ki of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-Subtilisin molecules that are activated later by those that are activated earlier.
