Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Mitsutoshi Toyama, Mitsuo Yamashita, Morihide Yoneda, Andrzej Zaborowski, Masaki Nagato, Hisayo Ono, Noriaki Hirayama, Yoshikatsu Murooka

研究成果: Article

14 引用 (Scopus)

抄録

Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and Brevibacterium. Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (kcat/Km) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high kcat value and a low Km value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.

元の言語English
ページ(範囲)477-483
ページ数7
ジャーナルProtein Engineering
15
発行部数6
出版物ステータスPublished - 2002
外部発表Yes

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Cholesterol Oxidase
Mutagenesis
Cholesterol
Enzymes
Substrates
Pregnenolone
Substitution reactions
Amino acids
Amino Acids
Catalyst activity
Oxidoreductases
Enzyme activity
Sterols

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

これを引用

Toyama, M., Yamashita, M., Yoneda, M., Zaborowski, A., Nagato, M., Ono, H., ... Murooka, Y. (2002). Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis. Protein Engineering, 15(6), 477-483.

Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis. / Toyama, Mitsutoshi; Yamashita, Mitsuo; Yoneda, Morihide; Zaborowski, Andrzej; Nagato, Masaki; Ono, Hisayo; Hirayama, Noriaki; Murooka, Yoshikatsu.

:: Protein Engineering, 巻 15, 番号 6, 2002, p. 477-483.

研究成果: Article

Toyama, M, Yamashita, M, Yoneda, M, Zaborowski, A, Nagato, M, Ono, H, Hirayama, N & Murooka, Y 2002, 'Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis', Protein Engineering, 巻. 15, 番号 6, pp. 477-483.
Toyama, Mitsutoshi ; Yamashita, Mitsuo ; Yoneda, Morihide ; Zaborowski, Andrzej ; Nagato, Masaki ; Ono, Hisayo ; Hirayama, Noriaki ; Murooka, Yoshikatsu. / Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis. :: Protein Engineering. 2002 ; 巻 15, 番号 6. pp. 477-483.
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abstract = "Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and Brevibacterium. Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (kcat/Km) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high kcat value and a low Km value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.",
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T1 - Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

AU - Toyama, Mitsutoshi

AU - Yamashita, Mitsuo

AU - Yoneda, Morihide

AU - Zaborowski, Andrzej

AU - Nagato, Masaki

AU - Ono, Hisayo

AU - Hirayama, Noriaki

AU - Murooka, Yoshikatsu

PY - 2002

Y1 - 2002

N2 - Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and Brevibacterium. Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (kcat/Km) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high kcat value and a low Km value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.

AB - Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and Brevibacterium. Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (kcat/Km) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high kcat value and a low Km value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.

KW - Cholesterol oxidase

KW - Site-directed mutagenesis

KW - Streptomyces

KW - Structural characterization

KW - Substrate specificity

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