Designer enzymes for glycosphingolipid synthesis by directed evolution

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.

When Enzymes Do It Better: Enzymatic Glycosylation Methods

“…Rhodococcus sp. endoglycoceraminidase [85,114]); and (iii) chemical complementation, in which a yeast three-hybrid system is used to link glycosynthase activity to the transcription of a reporter gene making cell growth dependent on glycosynthase product formation (e.g. Humicola insolens cellulase 7B [115,116].…”

“…Mutation of the catalytic nucleophile renders a glycosynthase able to use sialydated lactosyl fluoride donors to glycosylate sphingosine derivatives [84]. Further directed evolution of this enzyme has expanded the synthetic scope to different sphingoid acceptors [85,86]. Likewise, glycosylated small-molecule natural products are accessible by the glycosynthase technology.…”

Glycosynthases in Biocatalysis

“…that expanded the specificity of the GS toward phytosphingosine acceptor (see also below). [76] Conceivably, similar approaches can soon be adopted for the screening of more GS with novel specificities.…”

Toward Efficient Enzymatic Glycan Synthesis: Directed Evolution and Enzyme Engineering

“…This high-throughput screening methodology was applied to a library of EGC II glycosynthase mutants generated by error-prone PCR of the entire glycosynthase gene using the E351S variant as the parent template. [49] Ten thousand colonies were screened for their ability to transfer the oligosaccharyl moiety from G M1 OSF to either D-erythro-sphingosine or D-ribo-phytosphingosine. Several mutants with enhanced activity were identified from screening of the mutant library, including one in which a single mutation D314Y was further introduced to the E351S variant resulting in increased k cat for both G M1 OSF and D-erythro-sphingosine, and a several thousand-fold increase in k cat /K m for D-ribo-phytosphingosine to a first-order rate constant comparable to that of the parent enzyme for the native lipid acceptor D-erythro-sphingosine.…”