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Enzymatic engineering of modified Glycostructures

Objective

Oligosaccharides are a diverse class of carbohydrates widely distributed in Nature. Their biological significances range from roles as structural elements and energy sources to being ubiquitous constituents on mammalian, bacterial and viral cell surfaces, where they are involved in cell recognition events.

Both the selective modification of cell surface glycan structures and the production of competitive glycan ligands are important research areas with potential for developing novel therapeutics. The chemic al synthesis of oligosaccharides is a multi-step endeavour and is particularly difficult in analog synthesis, where additional steps are required to introduce modifications.

Increasingly chemists are employing enzymes to take advantage of their rate acceleration, stereoselectivity, regiospecificity and environmentally friendly green chemistry conditions. Our biocatalytic GlycoEngineering approach uses glycosyltransferases that catalyse the transfer of a monosaccharide from a nucleotide donor to an acceptor.

We have identified the blood group A and B synthesizing enzymes (GTA and GTB) as models for studying retaining glycosyltransferases. GTA and GTB will be employed for the synthesis of modified blood group glycostructures, using both enzyme engineering and substrate engineering.

Based on X-ray and NMR studies of ligand binding to GTA and GTB, different UDP-N-acetylgalactosamine and UDP-galactose donor analogs will be designed with docking and molecular modelling. To overcome potential limitations of native G TA and GTB in analog preparation, their substrate specificities will be broadened by mutagenesis based on sequence, structure and modelling studies.

Donor substrate analogs will be chemi-enzymatically prepared and used with native and mutated GTA and GTB t o synthesize modified oligosaccharide structures. This unique GlycoEngineering approach encompasses engineering both the enzymes and their substrates and can be broadly applied to other glycosyltransferases.

Call for proposal

FP6-2005-MOBILITY-5
See other projects for this call

Coordinator

CARLSBERG LABORATORY
Address
Gamle Carlsberg Vej 10
Copenhagen
Denmark