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Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase.
Casadevall, Guillem; Pierce, Colin; Guan, Bo; Iglesias-Fernandez, Javier; Lim, Huey-Yee; Greenberg, Lauren R; Walsh, Meghan E; Shi, Ke; Gordon, Wendy; Aihara, Hideki; Evans, Robert L; Kazlauskas, Romas; Osuna, Sílvia.
Afiliação
  • Casadevall G; Institut de Química Computacional i Catálisi and Departament de Química, Universitat de Girona, Carrer Maria Aurèlia Capmany 69, 17003 Girona, Spain.
  • Pierce C; Biotechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
  • Guan B; Biotechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
  • Iglesias-Fernandez J; Institut de Química Computacional i Catálisi and Departament de Química, Universitat de Girona, Carrer Maria Aurèlia Capmany 69, 17003 Girona, Spain.
  • Lim HY; Biotechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
  • Greenberg LR; Biotechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
  • Walsh ME; Biotechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
  • Shi K; Biotechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
  • Gordon W; Biotechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
  • Aihara H; Biotechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
  • Evans RL; Biotechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
  • Kazlauskas R; Biotechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 USA.
  • Osuna S; Institut de Química Computacional i Catálisi and Departament de Química, Universitat de Girona, Carrer Maria Aurèlia Capmany 69, 17003 Girona, Spain.
bioRxiv ; 2023 Aug 24.
Article em En | MEDLINE | ID: mdl-37662272
Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Tipo de estudo: Prognostic_studies / Risk_factors_studies Idioma: En Revista: BioRxiv Ano de publicação: 2023 Tipo de documento: Article País de afiliação: Espanha País de publicação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Tipo de estudo: Prognostic_studies / Risk_factors_studies Idioma: En Revista: BioRxiv Ano de publicação: 2023 Tipo de documento: Article País de afiliação: Espanha País de publicação: Estados Unidos