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A Mutant D-Fructose-6-Phosphate Aldolase (Ala129Ser) with Improved Affinity towards Dihydroxyacetone for the Synthesis of Polyhydroxylated Compounds

Authors

  • José A. Castillo,

    1. Laboratoire SEESIB, Clermont Université, Université Blaise Pascal, 24 avenue des Landais, 63177 Aubière cedex, France, Fax: (+33)-(0)4-7340-7717; phone: (+33)-(0)4-7340-7584
    2. CNRS, UMR 6504, 24 avenue des Landais, 63177 Aubière, France
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  • Christine Guérard-Hélaine,

    1. Laboratoire SEESIB, Clermont Université, Université Blaise Pascal, 24 avenue des Landais, 63177 Aubière cedex, France, Fax: (+33)-(0)4-7340-7717; phone: (+33)-(0)4-7340-7584
    2. CNRS, UMR 6504, 24 avenue des Landais, 63177 Aubière, France
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  • Mariana Gutiérrez,

    1. Biotransformation and Bioactive Molecules Group, Instituto de Química Avanzada de Cataluña – CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
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  • Xavier Garrabou,

    1. Biotransformation and Bioactive Molecules Group, Instituto de Química Avanzada de Cataluña – CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
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  • Martine Sancelme,

    1. Laboratoire SEESIB, Clermont Université, Université Blaise Pascal, 24 avenue des Landais, 63177 Aubière cedex, France, Fax: (+33)-(0)4-7340-7717; phone: (+33)-(0)4-7340-7584
    2. CNRS, UMR 6504, 24 avenue des Landais, 63177 Aubière, France
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  • Melanie Schürmann,

    1. Institute for Biotechnology 1, Forschungszentrum Jülich, 52405 Jülich, Germany
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  • Tomoyuki Inoue,

    1. Present address: Institute of Microbiology, Universität Stuttgart, Allmandring 31, 70550 Stuttgart, Germany, Fax: (+49)-(0)711-685-65725; phone: (+49)-(0)711-685-65488
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  • Virgil Hélaine,

    1. Laboratoire SEESIB, Clermont Université, Université Blaise Pascal, 24 avenue des Landais, 63177 Aubière cedex, France, Fax: (+33)-(0)4-7340-7717; phone: (+33)-(0)4-7340-7584
    2. CNRS, UMR 6504, 24 avenue des Landais, 63177 Aubière, France
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  • Franck Charmantray,

    1. Laboratoire SEESIB, Clermont Université, Université Blaise Pascal, 24 avenue des Landais, 63177 Aubière cedex, France, Fax: (+33)-(0)4-7340-7717; phone: (+33)-(0)4-7340-7584
    2. CNRS, UMR 6504, 24 avenue des Landais, 63177 Aubière, France
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  • Thierry Gefflaut,

    1. Laboratoire SEESIB, Clermont Université, Université Blaise Pascal, 24 avenue des Landais, 63177 Aubière cedex, France, Fax: (+33)-(0)4-7340-7717; phone: (+33)-(0)4-7340-7584
    2. CNRS, UMR 6504, 24 avenue des Landais, 63177 Aubière, France
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  • Laurence Hecquet,

    1. Laboratoire SEESIB, Clermont Université, Université Blaise Pascal, 24 avenue des Landais, 63177 Aubière cedex, France, Fax: (+33)-(0)4-7340-7717; phone: (+33)-(0)4-7340-7584
    2. CNRS, UMR 6504, 24 avenue des Landais, 63177 Aubière, France
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  • Jesús Joglar,

    1. Biotransformation and Bioactive Molecules Group, Instituto de Química Avanzada de Cataluña – CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
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  • Pere Clapés,

    1. Biotransformation and Bioactive Molecules Group, Instituto de Química Avanzada de Cataluña – CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
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  • Georg A. Sprenger,

    1. Present address: Institute of Microbiology, Universität Stuttgart, Allmandring 31, 70550 Stuttgart, Germany, Fax: (+49)-(0)711-685-65725; phone: (+49)-(0)711-685-65488
    2. Institute for Biotechnology 1, Forschungszentrum Jülich, 52405 Jülich, Germany
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  • Marielle Lemaire

    1. Laboratoire SEESIB, Clermont Université, Université Blaise Pascal, 24 avenue des Landais, 63177 Aubière cedex, France, Fax: (+33)-(0)4-7340-7717; phone: (+33)-(0)4-7340-7584
    2. CNRS, UMR 6504, 24 avenue des Landais, 63177 Aubière, France
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Abstract

A mutant of D-fructose-6-phosphate aldolase (FSA) of Escherichia coli, FSA A129S, with improved catalytic efficiency towards dihydroxyacetone (DHA), the donor substrate in aldol addition reactions, was explored for synthetic applications. The kcat/KM value for DHA was 17-fold higher with FSA A129S than that with FSA wild type (FSA wt). On the other hand, for hydroxyacetone as donor substrate FSA A129S was found to be 3.5-fold less efficient than FSA wt. Furthermore, FSA A129S also accepted glycolaldehyde (GA) as donor substrate with 3.3-fold lower affinity than FSA wt. This differential selectivity of both FSA wt and FSA A129S for GA makes them complementary biocatalysts allowing a control over donor and acceptor roles, which is particularly useful in carboligation multi-step cascade synthesis of polyhydroxylated complex compounds. Production of the mutant protein was also improved for its convenient use in synthesis. Several carbohydrates and nitrocyclitols were efficiently prepared, demonstrating the versatile potential of FSA A129S as biocatalyst in organic synthesis.

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