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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. | ỊFEBS Journal Function of conserved aromatic residues in the GaUGalNAc-glycosyltransferase motif of UDP-GalNAc polypeptide N-acetylgalactosaminyltransferase 1 Mari Tenno1 Aki Saeki1 Ake P. Elhammer2 and Akira Kurosaka1 1 Department of Biotechnology Faculty of Engineering Kyoto Sangyo University Japan 2 AureoGen Biosciences Inc. Kalamazoo MI USA Keywords aromatic residue kinetic analysis N-acetylgalactosaminyltransferase O-glycosylation site-directed mutagenesis Correspondence A. Kurosaka Department of Biotechnology Faculty of Engineering Kyoto Sangyo University Kamigamo-motoyama Kita-ku Kyoto 603-8555 Japan Fax 81 75 705 1914 Tel 81 75 705 1894 E-mail kurosaka@cc.kyoto-su.ac.jp Received 8 August 2007 revised 25 September 2007 accepted 2 October 2007 doi 10.1111 j.1742-4658.2007.06124.x 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
