TY - JOUR
T1 - Structural insights into the mechanism and inhibition of eukaryotic O-GlcNAc hydrolysis
AU - Rao, Francesco V.
AU - Dorfmueller, Helge C.
AU - Villa, Fabrizio
AU - Allwood, Matthew
AU - Eggleston, Ian M.
AU - Van Aalten, Daan M F
PY - 2006/4/5
Y1 - 2006/4/5
N2 - O-linked N-acetylglucosamine (O-GlcNAc) modification of specific serines/threonines on intracellular proteins in higher eukaryotes has been shown to directly regulate important processes such as the cell cycle, insulin sensitivity and transcription. The structure, molecular mechanisms of catalysis, protein substrate recognition/specificity of the eukaryotic O-GlcNAc transferase and hydrolase are largely unknown. Here we describe the crystal structure, enzymology and in vitro activity on human substrates of Clostridium perfringens NagJ, a close homologue of human O-GlcNAcase (OGA), representing the first family 84 glycoside hydrolase structure. The structure reveals a deep active site pocket highly conserved with the human enzyme, compatible with binding of O-GlcNAcylated peptides. Together with mutagenesis data, the structure supports a variant of the substrate-assisted catalytic mechanism, involving two aspartic acids and an unusually positioned tyrosine. Insights into recognition of substrate come from a complex with the transition state mimic O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (K i = 5.4 nM). Strikingly, the enzyme is inhibited by the pseudosubstrate peptide Ala-Cys(-S-GlcNAc)-Ala, and has OGA activity against O-GlcNAcylated human proteins, suggesting that the enzyme is a suitable model for further studies into the function of human OGA.
AB - O-linked N-acetylglucosamine (O-GlcNAc) modification of specific serines/threonines on intracellular proteins in higher eukaryotes has been shown to directly regulate important processes such as the cell cycle, insulin sensitivity and transcription. The structure, molecular mechanisms of catalysis, protein substrate recognition/specificity of the eukaryotic O-GlcNAc transferase and hydrolase are largely unknown. Here we describe the crystal structure, enzymology and in vitro activity on human substrates of Clostridium perfringens NagJ, a close homologue of human O-GlcNAcase (OGA), representing the first family 84 glycoside hydrolase structure. The structure reveals a deep active site pocket highly conserved with the human enzyme, compatible with binding of O-GlcNAcylated peptides. Together with mutagenesis data, the structure supports a variant of the substrate-assisted catalytic mechanism, involving two aspartic acids and an unusually positioned tyrosine. Insights into recognition of substrate come from a complex with the transition state mimic O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (K i = 5.4 nM). Strikingly, the enzyme is inhibited by the pseudosubstrate peptide Ala-Cys(-S-GlcNAc)-Ala, and has OGA activity against O-GlcNAcylated human proteins, suggesting that the enzyme is a suitable model for further studies into the function of human OGA.
KW - Glycosylation
KW - O-GlcNAc
KW - Phosphorylation
KW - Protein structure
KW - PUGNAc
UR - http://www.scopus.com/inward/record.url?scp=33645735070&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33645735070&partnerID=8YFLogxK
U2 - 10.1038/sj.emboj.7601026
DO - 10.1038/sj.emboj.7601026
M3 - Article
C2 - 16541109
AN - SCOPUS:33645735070
VL - 25
SP - 1569
EP - 1578
JO - EMBO Journal
JF - EMBO Journal
SN - 0261-4189
IS - 7
ER -