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Tricking Glycan Synthesis

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Glycan biosynthesis is primarily mediated by glycosyltransferases, a very large family of enzymes that contribute to the production of complex carbohydrates by sequentially adding monosaccharides to specific acceptor sugars. As might be expected from the complex array of glycan structures found in nature, glycosyltransferases have widely different specificities. The ability to harness the activity of these enzymes to particular substrates is an important goal for researchers working on the synthesis of new carbohydrate sequences.

Despite recent advances in carbohydrate engineering, until now, it was not possible to screen large libraries of glycosyltransferases as the reaction product does not result in measurable changes in absorbance or fluorescence. In an article in Nature Methods, Withers and colleagues describe a new fluorescence-based high-throughput screening approach for the directed evolution of CstII,  a sialyltransferase from the foodborne pathogen Campylobacter jejuni that catalyses the transfer of sialic acid (SA) to -galactose.

The authors introduced CstII mutant libraries along with fluorescently labeled galactose-containing acceptors and cytidine-5'-monophospho-N-acetyl-neuraminic acid (CMP-NeuAc) synthetase — to ensure the availability of the donor —  into an E. coli strain in which the degradation of SA and galactosidase is inhibited. Sialic acid is the only sugar that is charged under physiological conditions so the sialylated fluorescent product is retained in the cells after extensive washing. Finally, fluorescence-activated cell sorter (FACS) analysis was used to correlate the formation of transfer product with fluorescence levels in cells.

A screen of over 10⁶ CstII mutants revealed a new sialyltransferase variant with a much higher activity than the parent enzyme.  A single amino acid mutation, F91Y, appears to account for the dramatic increase in transfer activity. Structural analyses show that this mutation exposes a hydrophobic pocket that increases the affinity of the enzyme for the aromatic ring system of the biodipy dye used to label acceptors. Even though the activity of the CstII F91Y mutant towards unlabelled lactose and galactose sugars was not enhanced, this mutant can be used to transfer SA to biodipy-labeled acceptors that are not natural substrates of the wild-type enzyme.

In summary, the combination of a fluorescence-based assay with FACS has enabled for the first time high-throughput screening of sialyltransferase mutants for new catalytic activities. This method not only furthers our understanding of their mechanism of action, but also sheds new light on ways to direct glycan synthesis in vitro.

Author: Monica Hoyos Flight