Designer enzymes for glycosphingolipid synthesis  by directed evolution.
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.
Hancock
Susan M
SM
Department of Chemistry and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
Rich
Jamie R
JR
Caines
Matthew E C
ME
Strynadka
Natalie C J
NC
Withers
Stephen G
SG
D002384:Catalysis; D019020:Directed Molecular Evolution; D004797:Enzyme-Linked Immunosorbent Assay; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D006028:Glycosphingolipids / Q000138:chemical synthesis* / Q000737:chemistry; D017354:Point Mutation; D012240:Rhodococcus / Q000201:enzymology; D013379:Substrate Specificity
D006028:Glycosphingolipids; D006026:Glycoside Hydrolases