Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Cheriyan, M; Toone, EJ; Fierke, CA

Published Date

  • November 2007

Published In

Volume / Issue

  • 16 / 11

Start / End Page

  • 2368 - 2377

PubMed ID

  • 17962400

Pubmed Central ID

  • 17962400

Electronic International Standard Serial Number (EISSN)

  • 1469-896X

International Standard Serial Number (ISSN)

  • 0961-8368

Digital Object Identifier (DOI)

  • 10.1110/ps.073042907

Language

  • eng