The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform

Kohei Oda - One of the best experts on this subject based on the ideXlab platform.

  • Resynthesis of Reactive Site Peptide Bond and Temporary Inhibition of Streptomyces Metalloproteinase Inhibitor
    2016
    Co-Authors: Sailaja S. Seeram, Kazumi Hiraga, Kohei Oda
    Abstract:

    Streptomyces Metalloproteinase Inhibitor (SMPI) is a small proteinaceous Inhibitor which inhibits Metalloproteinases such as thermolysin (if, = 1.14X 10~10 M). When incubated with the enzyme, it is gradually hydrolyzed at the Cys64-Val65 peptide bond, which was identified as the reactive site by mutational analysis. To achieve a further understanding of the inhibition mechanism, we attempted to resynthesize the cleaved reactive site by using the enzyme catalytic action. The native Inhibitor was resynthesized from the modified in-hibitor (Ki =2.18X 10~8 M) by incubation with a catalytic amount of thermolysin under the same conditions as used for hydrolysis (pH 7.5, 25°C), suggesting that SMPI follows the standard mechanism of inhibition of serine proteinase Inhibitors. Temporary inhibition was observed when the native Inhibitor and thermolysin were incubated at a 1: 100 (mol/ mol) enzyme-Inhibitor ratio at 37°C. SMPI showed temporary inhibition towards all the enzymes it inhibited. The Inhibitory spectrum of SMPI was analyzed with various Metalloproteinases based on the Kt values and limited proteolysis patterns. Pseudomonas elastase and Streptomyces griseus Metalloproteinase II formed more stable complexes and showed much lower Kt values (~2 pM) than thermolysin. In the limited proteolysi

  • nmr structure of the streptomyces Metalloproteinase Inhibitor smpi isolated from streptomyces nigrescens tk 23 another example of an ancestral beta gamma crystallin precursor structure
    Journal of Molecular Biology, 1998
    Co-Authors: Ayako Ohno, Sailaja S. Seeram, Kazumi Hiraga, Kohei Oda, Shinichi Tate, Mark B Swindells, Masatsune Kainosho
    Abstract:

    Abstract The Streptomyces Metalloproteinase Inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous Metalloproteinase Inhibitor, and consists of 102 amino acid residues with two disulfide bridges. SMPI specifically inhibits Metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Cα, C′) r.m.s.d. values of 0.45(±0.11) A and 0.57(±0.18) A for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two β-sheets, each consisting of four antiparallel β-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key β-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the Inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain βγ-crystallin proteins, because of its structural similarity to each domain of the βγ-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the βγ-crystallin superfamily are believed to have evolved.

  • Identification of Reactive Site of a Proteinaceous Metalloproteinase Inhibitor from Streptomyces nigrescens TK-23
    Journal of biochemistry, 1997
    Co-Authors: Sailaja S. Seeram, Kazumi Hiraga, Atsushi Saji, Misao Tashiro, Kohei Oda
    Abstract:

    Streptomyces Metalloproteinase Inhibitor (SMPI), isolated from Streptomyces nigrescens TK-23, is a small proteinaceous Metalloproteinase Inhibitor consisting of 102 amino acid residues and two disulfide bridges. SMPI specifically inhibits Metalloproteinases such as thermolysin. After prolonged incubation with a catalytic amount of thermolysin, it is cleaved at Cys64-Val65 [Murai, H., Hara, S., Ikenaka, T., Oda, K., and Murao, S. (1985) J. Biochem. 97, 173-180]. Hence, for identification of the reactive site, mutants were constructed by substituting Val65 with various amino acid residues (Leu, Ile, Phe, Tyr, Gly, Ser, Lys, and Glu). The mutants were analyzed for Inhibitory activity. Among them, V65I, V65L, V65F, and V65Y retained strong Inhibitory activity, whereas V65S, V65G, V65K, and V65E showed very weak Inhibitory activity against thermolysin. The Ki values were found to be of the order of 10(10) M by using a fluorogenic substrate, MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. In addition, susceptibility to enzyme degradation was analyzed by means of limited proteolysis with thermolysin. Mutants which retained strong Inhibitory activity were cleaved by thermolysin only at the reactive site, in the same way as native SMPI. The mutants which showed weak Inhibitory activity underwent rapid degradation. These results were consistent with the substrate specificity of thermolysin. Based on these results, the reactive site of SMPI was identified as Cys64-Val65.

Sailaja S. Seeram - One of the best experts on this subject based on the ideXlab platform.

  • Resynthesis of Reactive Site Peptide Bond and Temporary Inhibition of Streptomyces Metalloproteinase Inhibitor
    2016
    Co-Authors: Sailaja S. Seeram, Kazumi Hiraga, Kohei Oda
    Abstract:

    Streptomyces Metalloproteinase Inhibitor (SMPI) is a small proteinaceous Inhibitor which inhibits Metalloproteinases such as thermolysin (if, = 1.14X 10~10 M). When incubated with the enzyme, it is gradually hydrolyzed at the Cys64-Val65 peptide bond, which was identified as the reactive site by mutational analysis. To achieve a further understanding of the inhibition mechanism, we attempted to resynthesize the cleaved reactive site by using the enzyme catalytic action. The native Inhibitor was resynthesized from the modified in-hibitor (Ki =2.18X 10~8 M) by incubation with a catalytic amount of thermolysin under the same conditions as used for hydrolysis (pH 7.5, 25°C), suggesting that SMPI follows the standard mechanism of inhibition of serine proteinase Inhibitors. Temporary inhibition was observed when the native Inhibitor and thermolysin were incubated at a 1: 100 (mol/ mol) enzyme-Inhibitor ratio at 37°C. SMPI showed temporary inhibition towards all the enzymes it inhibited. The Inhibitory spectrum of SMPI was analyzed with various Metalloproteinases based on the Kt values and limited proteolysis patterns. Pseudomonas elastase and Streptomyces griseus Metalloproteinase II formed more stable complexes and showed much lower Kt values (~2 pM) than thermolysin. In the limited proteolysi

  • nmr structure of the streptomyces Metalloproteinase Inhibitor smpi isolated from streptomyces nigrescens tk 23 another example of an ancestral beta gamma crystallin precursor structure
    Journal of Molecular Biology, 1998
    Co-Authors: Ayako Ohno, Sailaja S. Seeram, Kazumi Hiraga, Kohei Oda, Shinichi Tate, Mark B Swindells, Masatsune Kainosho
    Abstract:

    Abstract The Streptomyces Metalloproteinase Inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous Metalloproteinase Inhibitor, and consists of 102 amino acid residues with two disulfide bridges. SMPI specifically inhibits Metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Cα, C′) r.m.s.d. values of 0.45(±0.11) A and 0.57(±0.18) A for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two β-sheets, each consisting of four antiparallel β-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key β-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the Inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain βγ-crystallin proteins, because of its structural similarity to each domain of the βγ-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the βγ-crystallin superfamily are believed to have evolved.

  • Identification of Reactive Site of a Proteinaceous Metalloproteinase Inhibitor from Streptomyces nigrescens TK-23
    Journal of biochemistry, 1997
    Co-Authors: Sailaja S. Seeram, Kazumi Hiraga, Atsushi Saji, Misao Tashiro, Kohei Oda
    Abstract:

    Streptomyces Metalloproteinase Inhibitor (SMPI), isolated from Streptomyces nigrescens TK-23, is a small proteinaceous Metalloproteinase Inhibitor consisting of 102 amino acid residues and two disulfide bridges. SMPI specifically inhibits Metalloproteinases such as thermolysin. After prolonged incubation with a catalytic amount of thermolysin, it is cleaved at Cys64-Val65 [Murai, H., Hara, S., Ikenaka, T., Oda, K., and Murao, S. (1985) J. Biochem. 97, 173-180]. Hence, for identification of the reactive site, mutants were constructed by substituting Val65 with various amino acid residues (Leu, Ile, Phe, Tyr, Gly, Ser, Lys, and Glu). The mutants were analyzed for Inhibitory activity. Among them, V65I, V65L, V65F, and V65Y retained strong Inhibitory activity, whereas V65S, V65G, V65K, and V65E showed very weak Inhibitory activity against thermolysin. The Ki values were found to be of the order of 10(10) M by using a fluorogenic substrate, MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. In addition, susceptibility to enzyme degradation was analyzed by means of limited proteolysis with thermolysin. Mutants which retained strong Inhibitory activity were cleaved by thermolysin only at the reactive site, in the same way as native SMPI. The mutants which showed weak Inhibitory activity underwent rapid degradation. These results were consistent with the substrate specificity of thermolysin. Based on these results, the reactive site of SMPI was identified as Cys64-Val65.

Kazumi Hiraga - One of the best experts on this subject based on the ideXlab platform.

  • Resynthesis of Reactive Site Peptide Bond and Temporary Inhibition of Streptomyces Metalloproteinase Inhibitor
    2016
    Co-Authors: Sailaja S. Seeram, Kazumi Hiraga, Kohei Oda
    Abstract:

    Streptomyces Metalloproteinase Inhibitor (SMPI) is a small proteinaceous Inhibitor which inhibits Metalloproteinases such as thermolysin (if, = 1.14X 10~10 M). When incubated with the enzyme, it is gradually hydrolyzed at the Cys64-Val65 peptide bond, which was identified as the reactive site by mutational analysis. To achieve a further understanding of the inhibition mechanism, we attempted to resynthesize the cleaved reactive site by using the enzyme catalytic action. The native Inhibitor was resynthesized from the modified in-hibitor (Ki =2.18X 10~8 M) by incubation with a catalytic amount of thermolysin under the same conditions as used for hydrolysis (pH 7.5, 25°C), suggesting that SMPI follows the standard mechanism of inhibition of serine proteinase Inhibitors. Temporary inhibition was observed when the native Inhibitor and thermolysin were incubated at a 1: 100 (mol/ mol) enzyme-Inhibitor ratio at 37°C. SMPI showed temporary inhibition towards all the enzymes it inhibited. The Inhibitory spectrum of SMPI was analyzed with various Metalloproteinases based on the Kt values and limited proteolysis patterns. Pseudomonas elastase and Streptomyces griseus Metalloproteinase II formed more stable complexes and showed much lower Kt values (~2 pM) than thermolysin. In the limited proteolysi

  • nmr structure of the streptomyces Metalloproteinase Inhibitor smpi isolated from streptomyces nigrescens tk 23 another example of an ancestral beta gamma crystallin precursor structure
    Journal of Molecular Biology, 1998
    Co-Authors: Ayako Ohno, Sailaja S. Seeram, Kazumi Hiraga, Kohei Oda, Shinichi Tate, Mark B Swindells, Masatsune Kainosho
    Abstract:

    Abstract The Streptomyces Metalloproteinase Inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous Metalloproteinase Inhibitor, and consists of 102 amino acid residues with two disulfide bridges. SMPI specifically inhibits Metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Cα, C′) r.m.s.d. values of 0.45(±0.11) A and 0.57(±0.18) A for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two β-sheets, each consisting of four antiparallel β-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key β-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the Inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain βγ-crystallin proteins, because of its structural similarity to each domain of the βγ-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the βγ-crystallin superfamily are believed to have evolved.

  • Identification of Reactive Site of a Proteinaceous Metalloproteinase Inhibitor from Streptomyces nigrescens TK-23
    Journal of biochemistry, 1997
    Co-Authors: Sailaja S. Seeram, Kazumi Hiraga, Atsushi Saji, Misao Tashiro, Kohei Oda
    Abstract:

    Streptomyces Metalloproteinase Inhibitor (SMPI), isolated from Streptomyces nigrescens TK-23, is a small proteinaceous Metalloproteinase Inhibitor consisting of 102 amino acid residues and two disulfide bridges. SMPI specifically inhibits Metalloproteinases such as thermolysin. After prolonged incubation with a catalytic amount of thermolysin, it is cleaved at Cys64-Val65 [Murai, H., Hara, S., Ikenaka, T., Oda, K., and Murao, S. (1985) J. Biochem. 97, 173-180]. Hence, for identification of the reactive site, mutants were constructed by substituting Val65 with various amino acid residues (Leu, Ile, Phe, Tyr, Gly, Ser, Lys, and Glu). The mutants were analyzed for Inhibitory activity. Among them, V65I, V65L, V65F, and V65Y retained strong Inhibitory activity, whereas V65S, V65G, V65K, and V65E showed very weak Inhibitory activity against thermolysin. The Ki values were found to be of the order of 10(10) M by using a fluorogenic substrate, MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. In addition, susceptibility to enzyme degradation was analyzed by means of limited proteolysis with thermolysin. Mutants which retained strong Inhibitory activity were cleaved by thermolysin only at the reactive site, in the same way as native SMPI. The mutants which showed weak Inhibitory activity underwent rapid degradation. These results were consistent with the substrate specificity of thermolysin. Based on these results, the reactive site of SMPI was identified as Cys64-Val65.

Masatsune Kainosho - One of the best experts on this subject based on the ideXlab platform.

  • nmr structure of the streptomyces Metalloproteinase Inhibitor smpi isolated from streptomyces nigrescens tk 23 another example of an ancestral beta gamma crystallin precursor structure
    Journal of Molecular Biology, 1998
    Co-Authors: Ayako Ohno, Sailaja S. Seeram, Kazumi Hiraga, Kohei Oda, Shinichi Tate, Mark B Swindells, Masatsune Kainosho
    Abstract:

    Abstract The Streptomyces Metalloproteinase Inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous Metalloproteinase Inhibitor, and consists of 102 amino acid residues with two disulfide bridges. SMPI specifically inhibits Metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Cα, C′) r.m.s.d. values of 0.45(±0.11) A and 0.57(±0.18) A for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two β-sheets, each consisting of four antiparallel β-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key β-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the Inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain βγ-crystallin proteins, because of its structural similarity to each domain of the βγ-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the βγ-crystallin superfamily are believed to have evolved.

Danuta Prokopowiczi - One of the best experts on this subject based on the ideXlab platform.