In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.

中文翻译：

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盘基网柄菌和弓形虫缺氧感应脯氨酰羟化酶的生化和生物物理分析。


在动物中，对慢性缺氧的反应是由脯氨酰羟化酶 (PHD) 介导的，脯氨酰羟化酶 (PHD) 调节缺氧诱导转录因子 α (HIFα) 的水平。 PHD 同系物存在于其他类型的真核生物和原核生物中，它们作用于非 HIF 底物。为了深入了解不同 PHD 底物和特性的潜在因素，我们对来自细胞粘菌、盘基网柄菌和原生动物寄生虫、弓形虫（两者均缺乏 HIF）的 PHD 同源物进行了生化和生物物理研究。各自的脯氨酰羟化酶（DdPhyA 和 TgPhyA）催化 S 期激酶相关蛋白 1 (Skp1) 的脯氨酰羟化，该反应能够适应不同的双氧可用性。对全长 Skp1 底物的分析揭示了 DdPhyA 和 TgPhyA 的动力学特性存在显着差异，无论是彼此之间还是与人类 PHD2 相比；与细胞研究一致，TgPhyA 在低双氧浓度下比 DdPhyA 更活跃。 TgSkp1 是 DdPhyA 底物，DdSkp1 是 TgPhyA 底物。 DdPhyA/TgPhyA 底物与人 PHD2 之间未检测到交叉反应。人类 Skp1 E147P 变体是 DdPhyA 和 TgPhyA 底物，表明保留了一些祖先相互作用。 DdPhyA 的晶体学分析可以与人类、Trichoplax adhaerens 和原核生物的同源物进行比较，了解参与底物结合和催化的移动元件的差异。在 DdPhyA 中，PHD 中包围底物的两个移动环是保守的，但 PHD 的 C 端螺旋却明显缺失。综合结果支持这样的观点：PHD 同系物已经进化出适合其特定传感作用的动力学和结构特征。