Article abstract
Nature Chemical Biology 5, 508 - 514 (2009)
Published online: 14 June 2009 | doi:10.1038/nchembio.191
Designer enzymes for glycosphingolipid synthesis by directed evolution
Susan M Hancock1, Jamie R Rich1, Matthew E C Caines1, Natalie C J Strynadka1 & Stephen G Withers1
Abstract
Though glycosphingolipids have great potential as therapeutics for cancer, HIV, neurodegenerative diseases and auto-immune diseases, both extensive study of their biological roles and development as pharmaceuticals are limited by difficulties in their synthesis, especially on large scales. Here we addressed this restriction by expanding the synthetic scope of a glycosphingolipid-synthesizing enzyme through a combination of rational mutagenesis and directed evolution with an ELISA-based screening strategy. We targeted both a low-level promiscuous substrate activity and the overall catalytic efficiency of the catalyst, and we identified several mutants with enhanced activities. These new catalysts, which are capable of producing a broad range of homogeneous samples, represent a significant advance toward the facile, large-scale synthesis of glycosphingolipids and demonstrate the general utility of this approach toward the creation of designer glycosphingolipid-synthesizing enzymes.
- Department of Chemistry and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
Correspondence to: Stephen G Withers1 e-mail: withers@chem.ubc.ca
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