Probing Substrate Promiscuity of Amylosucrase from Neisseria polysaccharea

Authors

  • David Daudé,

    1. Université de Toulouse, INSA,UPS INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France, CNRS, UMR5504, F-31400 Toulouse, France, INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, Fax: (+33) 561559400
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  • Dr. Elise Champion,

    1. Université de Toulouse, INSA,UPS INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France, CNRS, UMR5504, F-31400 Toulouse, France, INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, Fax: (+33) 561559400
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  • Dr. Sandrine Morel,

    1. Université de Toulouse, INSA,UPS INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France, CNRS, UMR5504, F-31400 Toulouse, France, INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, Fax: (+33) 561559400
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  • Dr. David Guieysse,

    1. Université de Toulouse, INSA,UPS INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France, CNRS, UMR5504, F-31400 Toulouse, France, INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, Fax: (+33) 561559400
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  • Prof. Magali Remaud-Siméon,

    1. Université de Toulouse, INSA,UPS INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France, CNRS, UMR5504, F-31400 Toulouse, France, INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, Fax: (+33) 561559400
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  • Dr. Isabelle André

    Corresponding author
    1. Université de Toulouse, INSA,UPS INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France, CNRS, UMR5504, F-31400 Toulouse, France, INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, Fax: (+33) 561559400
    • Université de Toulouse, INSA,UPS INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France, CNRS, UMR5504, F-31400 Toulouse, France, INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, Fax: (+33) 561559400
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Abstract

The amylosucrase from Neisseria polysaccharea (NpAS) naturally catalyzes the synthesis of a variety of products from sucrose and shows signs of plasticity of its active site. To explore further this promiscuity, the tolerance of amylosucrase towards different donor and acceptor substrates was investigated. The selection of alternate donor substrates was first made on the basis of preliminary molecular modeling studies. From 11 potential donors harboring selective derivatizations that were experimentally evaluated, only p-nitrophenyl-α-D-glucopyranoside was used by the wild-type enzyme, and this underlines the high specificity of the −1 subsite of NpAS for glucosyl donor substrates. The acceptor substrate promiscuity was further explored by screening 20 hydroxylated molecules, including D- and L-monosaccharides as well as polyols. With the exception of one compound, all were successfully glucosylated, and this showcases the tremendous plasticity of the +1 subsite of NpAS, which is responsible for acceptor recognition. The products obtained from the transglucosylation reactions of three selected acceptors were characterized, and they revealed original structures and enzyme enantiopreference, which were more particularly analyzed by in silico docking analyses.

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