Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli.
The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H-precursor structure Fuc alpha (1-2)Gal beta-OR to form the blood group A and B antigens, respectively. Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase. A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed in Escherichia coli. The purified hybrid glycosyltransferases were characterized by two-substrate enzyme kinetic analysis using both UDP-GalNAc and UDP-Gal donor substrates. The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP-GlcNAc and UDP-Glc, respectively. The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity.
Seto
N O
NO
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada. nina.seto@nrc.ca
Compston
C A
CA
Evans
S V
SV
Bundle
D R
DR
Narang
S A
SA
Palcic
M M
MM
D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000235:genetics*; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D013379:Substrate Specificity; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014531:Uridine Diphosphate Galactose; D014536:Uridine Diphosphate N-Acetylgalactosamine; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase; C023155:blood-group-substance alpha-D-galactosyltransferase