Rapid screening of sugar-nucleotide donor specificities of putative glycosyltransferases.
Determining the correct enzymatic activity of putative glycosyltransferases (GTs) can be challenging as these enzymes can utilize multiple donor and acceptor substrates. Upon initial determination of the donor-sugar nucleotide(s), a GT utilizes various acceptor molecules that can then be tested. Here, we describe a quick method to screen sugar-nucleotide donor specificities of GTs utilizing a sensitive, nonradioactive, commercially available bioluminescent uridine diphosphate detection kit. This in vitro method allowed us to validate the sugar-nucleotide donor-substrate specificities of recombinantly expressed human, bovine, bacterial and protozoan GTs. Our approach, which is less time consuming than many traditional assays that utilize radiolabeled sugars and chromatographic separations, should facilitate discovery of novel GTs that participate in diverse biological processes.
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.
M Osman
MO
Sheikh
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.|Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
Stephanie M
SM
Halmo
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.|Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
Sneha
S
Patel
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.|Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.|Center for Molecular Medicine, University of Georgia, Athens, GA, USA.
Dustin
D
Middleton
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.|Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
Hideyuki
H
Takeuchi
Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.
Christopher M
CM
Schafer
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
Christopher M
CM
West
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.|Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
Robert S
RS
Haltiwanger
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.|Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.|Center for Molecular Medicine, University of Georgia, Athens, GA, USA.
Fikri Y
FY
Avci
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.|Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
Kelley W
KW
Moremen
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.|Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
Lance
L
Wells
D000818:Animals; D001419:Bacteria / Q000201:enzymology; D002417:Cattle; D016695:Glycosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D009711:Nucleotides / Q000737:chemistry*; D013379:Substrate Specificity; D000073893:Sugars / Q000737:chemistry*
D009711:Nucleotides; D000073893:Sugars; D016695:Glycosyltransferases
assays; enzymology; glycosyltransferase; methods; sugar-nucleotide