New Structural Motif for Carboxylic Acid Perhydrolases

Authors

  • DeLu (Tyler) Yin,

    1. University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics, and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 (USA)
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  • Vince M. Purpero,

    1. University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics, and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 (USA)
    2. Current address: Lucigen Corp., 2120 West Greenview Dr. Middleton, WI 53562 (USA)
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  • Ryota Fujii,

    1. University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics, and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 (USA)
    2. Current address: Mitsui Chemicals Singapore R&D Centre, 50 Science Park Road, #06-08, The Kendall, Singapore Science Park II, Singapore 117406 (Singapore)
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  • Qing Jing,

    1. University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics, and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 (USA)
    2. Current address: Northwestern University, Department of Chemistry, 2145 Sheridan Road, Evanston, IL 60208 (USA)
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  • Romas J. Kazlauskas

    Corresponding author
    1. University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics, and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 (USA)
    • University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics, and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 (USA)
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Abstract

Some serine hydrolases also catalyze a promiscuous reaction— reversible perhydrolysis of carboxylic acids to make peroxycarboxylic acids. Five X-ray crystal structures of these carboxylic acid perhydrolases show a proline in the oxyanion loop. Here, we test whether this proline is essential for high perhydrolysis activity using Pseudomonas fluorescens esterase (PFE). The L29P variant of this esterase catalyzes perhydrolysis 43-fold faster (kcat comparison) than the wild type. Surprisingly, saturation mutagenesis at the 29 position of PFE identified six other amino acid substitutions that increase perhydrolysis of acetic acid at least fourfold over the wild type. The best variant, L29I PFE, catalyzed perhydrolysis 83-times faster (kcat comparison) than wild-type PFE and twice as fast as L29P PFE. Despite the different amino acid in the oxyanion loop, L29I PFE shows a similar selectivity for hydrogen peroxide over water as L29P PFE (β0=170 vs. 160 M−1), and a similar fast formation of acetyl-enzyme (140 vs. 62 U mg−1). X-ray crystal structures of L29I PFE with and without bound acetate show an unusual mixture of two different oxyanion loop conformations. The type II β-turn conformation resembles the wild-type structure and is unlikely to increase perhydrolysis, but the type I β-turn conformation creates a binding site for a second acetate. Modeling suggests that a previously proposed mechanism for L29P PFE can be extended to include L29I PFE, so that an acetate accepts a hydrogen bond to promote faster formation of the acetyl-enzyme.

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