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Protein Science (2007), 16:2368-2377. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates

Manoj Cheriyan1, Eric J. Toone2, and Carol A. Fierke1

1 Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA
2 Department of Chemistry, Duke University, Durham, North Carolina 27708, USA

(RECEIVED May 30, 2007; FINAL REVISION August 10, 2007; ACCEPTED August 10, 2007)

Narrow substrate specificities often limit the use of enzymes in biocatalysis. To further the development of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase as a biocatalyst, the molecular determinants of substrate specificity were probed by mutagenesis. Our data demonstrate that S184 is located in the substrate-binding pocket and interacts with the phosphate moiety of KDPG, providing biochemical support for the binding model proposed on the basis of crystallographic data. An analysis of the substrate selectivity of the mutant enzymes indicates that alterations to the phosphate-binding site of KDPG aldolase changes the substrate selectivity. We report mutations that enhance catalysis of aldol cleavage of substrates lacking a phosphate moiety and demonstrate that electrophile reactivity correlates with the hydrophobicity of the substituted side chain. These mutations improve the selectivity for unnatural substrates as compared to KDPG by up to 2000-fold. Furthermore, the S184L KDPG aldolase mutant improves the catalytic efficiency for the synthesis of a precursor for nikkomycin by 40-fold, making it a useful biocatalyst for the preparation of fine chemicals.

Keywords: nikkomycin; aldolase; KDPG; substrate specificity; rational redesign; active site; directed evolution; protein engineering; biocatalysis


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