Vertebrates use the mannose 6-phosphate (M6P)-recognition system to deliver lysosomal hydrolases to lysosomes. Key to this pathway is N-acetylglucosamine (GlcNAc)-1-phosphotransferase (PTase) that selectively adds GlcNAc-phosphate (P) to mannose residues of hydrolases. Human PTase is an α₂β₂γ₂ heterohexamer with a catalytic core and several peripheral domains that recognize and bind substrates. Here we report a cryo-EM structure of the catalytic core of human PTase and the identification of a hockey stick-like motif that controls activation of the enzyme. Movement of this motif out of the catalytic pocket is associated with a rearrangement of part of the peripheral domains that unblocks hydrolase glycan access to the catalytic site, thereby activating PTase. We propose that PTase fluctuates between inactive and active states in solution, and selective substrate binding of a lysosomal hydrolase through its protein-binding determinant to PTase locks the enzyme in the active state to permit glycan phosphorylation. This mechanism would help ensure that only N-linked glycans of lysosomal enzymes are phosphorylated.

脊椎动物使用甘露糖 6-磷酸 (M6P) 识别系统将溶酶体水解酶递送至溶酶体。该途径的关键是N-乙酰葡糖胺 (GlcNAc)-1-磷酸转移酶 (PTase)，它选择性地将 GlcNAc-磷酸 (P) 添加到水解酶的甘露糖残基上。人 PTase 是一种 α ₂ β ₂ γ ₂具有催化核心和几个识别和结合底物的外围结构域的异六聚体。在这里，我们报告了人类 PTase 催化核心的低温 EM 结构和控制酶激活的曲棍球棒状基序的鉴定。该基序从催化口袋中移出与部分外围结构域的重排有关，该结构域会阻止水解酶聚糖进入催化位点，从而激活 PTase。我们提出 PTase 在溶液中在非活性和活性状态之间波动，并且溶酶体水解酶通过其蛋白质结合决定簇与 PTase 的选择性底物结合将酶锁定在活性状态以允许聚糖磷酸化。这种机制将有助于确保只有N溶酶体酶的连接聚糖被磷酸化。