Mutational Analysis of the Catalytic Domain of O-Linked N-Acetylglucosaminyl Transferase

• Published in: The Journal of biological chemistry Vol. 280; no. 42; pp. 35537 - 35544
• Main Authors: Lazarus, Brooke D., Roos, Mark D., Hanover, John A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.10.2005; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Binding Sites; Catalytic Domain; DNA Mutational Analysis; Electrophoresis, Polyacrylamide Gel; Genetic Vectors; Glycosylation; Hexosamines - chemistry; Humans; Insulin Resistance; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; N-Acetylglucosaminyltransferases - chemistry; N-Acetylglucosaminyltransferases - metabolism; Nuclear Pore - chemistry; Nucleotides - chemistry; Point Mutation; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M504948200

O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT (CD I and CD II), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues (two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CD I and CD II, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades.