Location and characterization of the O-GlcNAcase active site

NCOAT is a bifunctional nucleo-cytoplasmic protein with both O-GIcNAcase and histone acetyltransferase domains. The O-GIcNAcase domain catalyzes the removal of O-linked GlcNAc modifications from proteins and we have found that it resides in the N-terminal third of NCOAT. The recognition of the substrate GIcNAc suggests that the O-GIcNAcase is related in structure and catalytic mechanism to chitinases, hexosaminidases and hyaluronidases. These families of glycosidases all possess a catalytic doublet of carboxylate-containing residues, with one providing an acid-base function, and the second acting to orient and use the N-acetyl group of GIcNAc during catalysis. Indeed, we show that the O-GIcNAcase also possesses the catalytic doublet motif shared among these enzymes and that these two essential residues are aspartic acids at positions 175 and 177, respectively, in mouse NCOAT. In addition, a conserved cysteine at 166 and a conserved aspartic acid at 174 were also found to be necessary for fully efficient enzymatic activity. Given this information, we propose that the O-GIcNAcasc active site resembles those of the above glycosidases which carry out the hydrolysis of GIcNAc linkages in a substrate-assisted acid-base manner. (c) 2006 Elsevier B.V. All rights reserved.