Endolysosomal exonucleases PLD3 and PLD4 (phospholipases D3 and D4) are associated with autoinflammatory and autoimmune diseases. We report structures of these enzymes, and the molecular basis of their catalysis. The structures reveal an intra-chain dimer topology forming a basic active site at the interface. Like other PLD superfamily members, PLD3 and PLD4 carry HxKxxxxD/E motifs and participate in phosphodiester-bond cleavage. The enzymes digest ssDNA and ssRNA in a 5′-to-3′ manner and are blocked by 5′-phosphorylation. We captured structures in apo, intermediate, and product states and revealed a “link-and-release” two-step catalysis. We also unexpectedly demonstrated phosphatase activity via a covalent 3-phosphohistidine intermediate. PLD4 contains an extra hydrophobic clamp that stabilizes substrate and could affect oligonucleotide substrate preference and product release. Biochemical and structural analysis of disease-associated mutants of PLD3/4 demonstrated reduced enzyme activity or thermostability and the possible basis for disease association. Furthermore, these findings provide insight into therapeutic design.

中文翻译：

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对疾病相关内溶酶体核酸外切酶 PLD3 和 PLD4 的结构和机制的见解

内溶酶体核酸外切酶 PLD3 和 PLD4（磷脂酶 D3 和 D4）与自身炎症和自身免疫性疾病相关。我们报告了这些酶的结构及其催化的分子基础。该结构揭示了链内二聚体拓扑结构，在界面处形成基本活性位点。与其他 PLD 超家族成员一样，PLD3 和 PLD4 携带 HxKxxxxD/E 基序并参与磷酸二酯键裂解。这些酶以 5' 至 3' 的方式消化 ssD​​NA 和 ssRNA，并被 5'-磷酸化阻断。我们捕获了 apo、中间体和产物状态的结构，并揭示了“连接和释放”两步催化。我们还出乎意料地通过共价 3-磷酸组氨酸中间体证明了磷酸酶活性。 PLD4 包含一个额外的疏水夹，可稳定底物并可能影响寡核苷酸底物偏好和产物释放。与疾病相关的 PLD3/4 突变体的生化和结构分析表明酶活性或热稳定性降低以及疾病关联的可能基础。此外，这些发现为治疗设计提供了见解。