Mutational analysis of the catalytic domain of O-linked N-acetylglucosaminyl transferase.
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.
Lazarus
Brooke D
BD
Laboratory of Cell Biology and Biochemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Roos
Mark D
MD
Hanover
John A
JA
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006031:Glycosylation; D006595:Hexosamines / Q000737:chemistry; D006801:Humans; D007333:Insulin Resistance; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D022022:Nuclear Pore / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D006595:Hexosamines; D009711:Nucleotides; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase