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Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory

Authors

  • Maria Svedendahl,

    1. Department of Biochemistry, Royal Institute of Technology (KTH), AlbaNova University Center, 106 91 Stockholm (Sweden), Fax: (+46) 8-5537-8483
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  • Peter Carlqvist Dr.,

    1. Physical Chemistry, Royal Institute of Technology (KTH), 100 44 Stockholm (Sweden), Fax: (+46) 8-790-8207
    2. Present address: Medivir AB, P. O. Box 1086, SE-14 122 Huddinge (Sweden)
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  • Cecilia Branneby Dr.,

    1. Department of Biochemistry, Royal Institute of Technology (KTH), AlbaNova University Center, 106 91 Stockholm (Sweden), Fax: (+46) 8-5537-8483
    2. Present address: BASF Aktiengesellschaft, Abt. GVF/E-A 30, 67056 Ludwigshafen (Germany)
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  • Olof Allnér,

    1. Physical Chemistry, Royal Institute of Technology (KTH), 100 44 Stockholm (Sweden), Fax: (+46) 8-790-8207
    2. Present address: Centre for Structural Biochemistry, Karolinska Institute, 14 157 Huddinge (Sweden)
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  • Anton Frise,

    1. Physical Chemistry, Royal Institute of Technology (KTH), 100 44 Stockholm (Sweden), Fax: (+46) 8-790-8207
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  • Karl Hult Prof.,

    1. Department of Biochemistry, Royal Institute of Technology (KTH), AlbaNova University Center, 106 91 Stockholm (Sweden), Fax: (+46) 8-5537-8483
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  • Per Berglund Prof.,

    1. Department of Biochemistry, Royal Institute of Technology (KTH), AlbaNova University Center, 106 91 Stockholm (Sweden), Fax: (+46) 8-5537-8483
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  • Tore Brinck Prof.

    1. Physical Chemistry, Royal Institute of Technology (KTH), 100 44 Stockholm (Sweden), Fax: (+46) 8-790-8207
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Abstract

Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of α,β-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated by density functional theory calculations, and it was demonstrated that the reaction proceeds through a two step-mechanism with formation of an oxyanionic intermediate. The active-site residue His224 functions as a general acid-base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.

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