Characterization of NCOAT, a bifunctional enzyme with O-GlcNAcase and histone acetyltransferase activity

O-GlcNAcylation is a ubiquitous post-translational modification whereby the monosaccharide N-acetylglucosamine (GlcNAc) is affixed via a beta linkage to specific serine or threonine hydroxyls on target proteins. These modifications can be selectively removed by the enzyme O-GlcNAcase which hydrolyzes the glycosidic bond and liberates the free protein and (GlcNAc) O-GlcNAcase recognizes the same common core substrate, GlcNAc, that chitinases, hexosaminidases, and hyaluronidases recognize and is therefore likely to exhibit a mode of catalysis and substrate binding remarkably similar to that of these enzymes, which have a highly conserved mechanism for these functions as well as conserved amino acids which carry out these events. This information, along with the use of naturally occurring splice variant isoforms, was used to find the active site of O-GlcNAcase and to elucidate amino acids necessary for efficient catalysis. Once demonstrated, this information was utilized to determine the mechanism by which the diabetogenic drug and selective O-GlcNAcase inhibitor streptozotocin can perform its inhibitory effects on the enzyme. Since streptozotocin is a GlcNAc analog, the enzyme will attempt to catalyze it as it would a normal substrate, converting streptozotocin to a transition-state analog that is more stable than the natural ligand transition-state is and therefore competes for the active site more expediently. Over the course of these studies, O-GlcNAcase was realized to have a domain in its C terminus with secondary structure similar to those of several different acetyltransferase active sites. O-GlcNAcase was found to possess acetyltransferase activity for specific target lysines in nucleosomal and free histone substrates, prompting us to rename the enzyme NCOAT, for nuclear and cytosolic O-GlcNAcase and acetyltransferase. The active site for this second activity was found to have secondary structure nearly identical to those previously characterized and had critical catalytic residues in positions corresponding to the counterpart enzymes.