Function of conserved aromatic residues in the Gal/GalNAc-glycosyltransferase motif of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1.
UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases), which initiate mucin-type O-glycan biosynthesis, have broad acceptor substrate specificities, and it is still unclear how they recognize peptides with different sequences. To increase our understanding of the catalytic mechanism of GalNAc-T1, one of the most ubiquitous isozymes, we studied the effect of substituting six conserved aromatic residues in the highly conserved Gal/GalNAc-glycosyltransferase motif with leucine on the catalytic properties of the enzyme. Our results indicate that substitutions of Trp302 and Phe325 have little impact on enzyme function and that substitutions of Phe303 and Tyr309 could be made with only limited impact on the interaction(s) with donor and/or acceptor substrates. By contrast, Trp328 and Trp316 are essential residues for enzyme functions, as substitution with leucine, at either site, led to complete inactivation of the enzymes. The roles of these tryptophan residues were further analyzed by evaluating the impact of substitutions with additional amino acids. All evaluated substitutions at Trp328 resulted in enzymes that were completely inactive, suggesting that the invariant Trp328 is essential for enzymatic activity. Trp316 mutant enzymes with nonaromatic replacements were again completely inactive, whereas two mutant enzymes containing a different aromatic amino acid, at position 316, showed low catalytic activity. Somewhat surprisingly, a kinetic analysis revealed that these two amino acid substitutions had a moderate impact on the enzyme's affinity for the donor substrate. By contrast, the drastically reduced affinity of the Trp316 mutant enzymes for the acceptor substrates suggests that Trp316 is important for this interaction.
Tenno
Mari
M
Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Japan.
Saeki
Aki
A
Elhammer
Ake P
AP
Kurosaka
Akira
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002417:Cattle; D002522:Chlorocebus aethiops; D018076:DNA, Complementary; D005752:Gastric Mucins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017350:N-Acetylgalactosaminyltransferases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013379:Substrate Specificity; D014530:Uridine Diphosphate / Q000737:chemistry*; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D024322:Amino Acids, Aromatic; D018076:DNA, Complementary; D005752:Gastric Mucins; D007527:Isoenzymes; D007930:Leucine; D017350:N-Acetylgalactosaminyltransferases; D011993:Recombinant Fusion Proteins; D014530:Uridine Diphosphate; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C053366:apomucin