Structure determination reveals the mechanistic basis of mannose-6-P signal generation by the dimeric lysosomal...

N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (NAGPA), also known as the uncovering enzyme, catalyzes the final step in mannose-6-phosphate signal generation for lysosomal enzyme trafficking. Despite its central role, the enzyme's architecture, catalytic mechanism, and the contribution of its C-terminal region remain incompletely defined. Here, we combine solution biophysics, cryo-EM, structural modeling, and a quantitative cell-based assay to characterize human NAGPA. We show that NAGPA forms a noncovalent dimer in solution, resolving prior uncertainty regarding disulfide-linked higher-order assemblies. The structure reveals an elongated dimer composed of two catalytic cores and two C-terminal epidermal growth factor-like stalks, semi-rigid in nature, that likely position the catalytic domains ∼5 nm from the membrane. A structure determined in the presence of the substrate analog GlcNAc-1-phosphate captures GlcNAc and phosphate in the active site, identifying an invariant DGGGS motif that is critical for substrate recognition and enzyme catalysis. Based on these observations, we propose a substrate-assisted S_Ni-like mechanism for cleavage of the glycosidic C-O bond between GlcNAc and mannose-6-phosphate. Functional assays show that the membrane-tethered full-length NAGPA is more active than the isolated catalytic core, and that mutations in the hinge linking the catalytic domain to the C-terminal stalk reduce activity. Together, these findings establish a structural and mechanistic framework for understanding NAGPA function in lysosomal enzyme targeting.