Methods for enzymatic conversion of GDP-mannose to GDP-fucose
AssigneeCytel Corporation
InventorEric R. Sjoberg
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Abstract
This invention provides methods for practical enzymatic conversion of GDP-mannose to GDP-fucose. These methods are useful for efficient synthesis of reactants used in the synthesis of fucosylated oligosaccharides.
Claims55
An expression vector that comprises a promoter operably linked to a nucleic acid that encodes a prokaryotic enzyme that has both an epimerase and a reductase activity that catalyze the conversion of GDP-4-keto-6-deoxymannose to GDP-fucose, wherein the expression vector lacks an E. coli wcaI coding region.
A reaction mixture for synthesizing GDP-fucose, the reaction mixture comprising GDP-4-keto-6-deoxymannose, NADPH, and a prokaryotic enzyme that has both an epimerase and a reductase activity, wherein the prokaryotic enzyme can catalyze the conversion of GDP-4-keto-6-deoxymannose to GDP-fucose.
A method for enzymatic conversion of GDP-mannose to GDP-fucose, the method comprising:
- a) providing a reaction mixture that comprises GDP-mannose, GDP-mannose 4,6-dehydratase, and NADP+;
- b) incubating the reaction mixture for a sufficient time to convert at least about 90% of the GDP-mannose to GDP-4-keto-6-deoxymannose;
- c) adding to the reaction mixture one or more polypeptides having GDP-4-keto-6-deoxymannose 3,5-epimerase and GDP-4-keto-6-galactose reductase activities;
- and
- d) incubating the reaction mixture for a sufficient time to convert the GDP-4-keto-6-deoxymannose to GDP-fucose.
A method for the preparation of a fucosylated oligosaccharide, the method comprising contacting an acceptor saccharide with a fucosylation reaction mixture that comprises GDP-fucose and a fucosyltransferase which transfers fucose from the GDP-fucose to provide said fucosylated oligosaccharide, wherein the efficiency of said fucosylation is enhanced by one or more efficiency-enhancing steps selected from the group consisting of:
- 1) forming said GDP-fucose by enzymatic conversion of GDP-mannose to GDP-fucose by:
- a) providing a reaction mixture that comprises GDP-mannose, GDP-mannose 4,6-dehydratase, and NADP+;
- b) incubating the reaction mixture for a sufficient time to convert at least about 90% of the GDP-mannose to GDP-4-keto-6-deoxymannose;
- c) adding to the product of step b) one or more polypeptides having GDP-4-keto-6-deoxymannose 3,5-epimerase and GDP-4-keto-6-galactose reductase activities;
- and
- d) incubating the reaction mixture for a sufficient time to convert the GDP-4-keto-6-deoxymannose to GDP-fucose;
- 2) adding pyruvate kinase and a substrate for the pyruvate kinase to the fucosylation reaction mixture, wherein GDP produced as a result of the transfer of fucose from the GDP-fucose is converted to GTP;
- and
- 3) conducting the fucosylation in a reaction medium that comprises a soluble divalent metal cation, wherein said medium is supplemented with said soluble divalent metal cation to maintain the concentration of said divalent metal cation between about 2 mM and about 75 mM.
Dependencies are inferred from the claim text.