Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes.
The human blood group A and B glycosyltransferase enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176 --> Gly, Gly-235 --> Ser, Leu-266 --> Met, and Gly-268 --> Ala) is sufficient to change the enzyme specificity from a blood group A to a blood group B glycosyltransferase. To carry out a systematic study, a synthetic gene strategy was employed to obtain their genes and to allow facile mutagenesis. Soluble forms of a recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically characterized extensively for the first time. A functional hybrid A/B mutant enzyme was able to catalyze both A and B reactions, with the kcat being 5-fold higher for the A donor. Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corresponding residue from glycosyltransferase B (Arg-176 --> Gly) produced enzymes with glycosyltransferase A activity only, but with very large (11-fold) increases in the kcat and increased specificity. The increases observed in kcat are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.
Seto
N O
NO
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6. nina.seto@nrc.ca
Palcic
M M
MM
Compston
C A
CA
Li
H
H
Bundle
D R
DR
Narang
S A
SA
D000017:ABO Blood-Group System; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity / Q000235:genetics
D000017:ABO Blood-Group System; D000596:Amino Acids; D011994:Recombinant Proteins; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase; C023155:blood-group-substance alpha-D-galactosyltransferase