Synthesis of 2-oxoglutarate derivatives and their evaluation as cosubstrates and inhibitors of human aspartate/asparagine-β-hydroxylase,Chemical Science

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Synthesis of 2-oxoglutarate derivatives and their evaluation as cosubstrates and inhibitors of human aspartate/asparagine-β-hydroxylase
Chemical Science ( IF 7.6 ) Pub Date : 2020-12-7 , DOI: 10.1039/d0sc04301j
Lennart Brewitz ₁ , Yu Nakashima ₁ , Christopher J Schofield ₁

Affiliation

2-Oxoglutarate (2OG) is involved in biological processes including oxidations catalyzed by 2OG oxygenases for which it is a cosubstrate. Eukaryotic 2OG oxygenases have roles in collagen biosynthesis, lipid metabolism, DNA/RNA modification, transcriptional regulation, and the hypoxic response. Aspartate/asparagine-β-hydroxylase (AspH) is a human 2OG oxygenase catalyzing post-translational hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs) in the endoplasmic reticulum. AspH is of chemical interest, because its Fe(II) cofactor is complexed by two rather than the typical three residues. AspH is upregulated in hypoxia and is a prognostic marker on the surface of cancer cells. We describe studies on how derivatives of its natural 2OG cosubstrate modulate AspH activity. An efficient synthesis of C3- and/or C4-substituted 2OG derivatives, proceeding via cyanosulfur ylid intermediates, is reported. Mass spectrometry-based AspH assays with >30 2OG derivatives reveal that some efficiently inhibit AspH via competing with 2OG as evidenced by crystallographic and solution analyses. Other 2OG derivatives can substitute for 2OG enabling substrate hydroxylation. The results show that subtle changes, e.g. methyl- to ethyl-substitution, can significantly alter the balance between catalysis and inhibition. 3-Methyl-2OG, a natural product present in human nutrition, was the most efficient alternative cosubstrate identified; crystallographic analyses reveal the binding mode of (R)-3-methyl-2OG and other 2OG derivatives to AspH and inform on the balance between turnover and inhibition. The results will enable the use of 2OG derivatives as mechanistic probes for other 2OG utilizing enzymes and suggest 2-oxoacids other than 2OG may be employed by some 2OG oxygenases in vivo.

中文翻译：

2-酮戊二酸衍生物的合成及其作为人天冬氨酸/天冬酰胺-β-羟化酶共底物和抑制剂的评价

2-氧戊二酸 (2OG) 参与生物过程，包括 2OG 加氧酶催化的氧化反应，它是 2OG 加氧酶的共底物。真核生物 2OG 加氧酶在胶原蛋白生物合成、脂质代谢、DNA/RNA 修饰、转录调节和缺氧反应中发挥作用。天冬氨酸/天冬酰胺-β-羟化酶 (AspH) 是一种人 2OG 加氧酶，催化内质网表皮生长因子样结构域 (EGFD) 中 Asp/Asn 残基的翻译后羟基化。 AspH 具有化学意义，因为它的 Fe( II ) 辅因子由两个而不是典型的三个残基络合。 AspH 在缺氧情况下上调，是癌细胞表面的预后标志物。我们描述了关于其天然 2OG 共底物的衍生物如何调节 AspH 活性的研究。据报道，通过氰基硫内鎓中间体进行 C3-和/或 C4-取代的 2OG 衍生物的有效合成。对 >30 2OG 衍生物进行的基于质谱的 AspH 测定表明，某些衍生物通过与 2OG 竞争来有效抑制 AspH，这一点已通过晶体学和溶液分析得到证实。其他 2OG 衍生物可以替代 2OG，实现底物羟基化。结果表明，细微的变化，例如甲基取代为乙基，可以显着改变催化和抑制之间的平衡。 3-甲基-2OG 是人类营养中存在的一种天然产物，是已确定的最有效的替代共底物；晶体学分析揭示了 ( R )-3-甲基-2OG 和其他 2OG 衍生物与 AspH 的结合模式，并揭示了周转和抑制之间的平衡。这些结果将使得能够使用2OG衍生物作为其他2OG利用酶的机械探针，并表明除了2OG之外的2-含氧酸可以被体内的一些2OG加氧酶使用。

更新日期：2020-12-09

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