Evidence that two enzyme-derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting HIF (FIH)
Evidence that two enzyme-derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting HIF (FIH)
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DOI：
10.1074/jbc.m804999200
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发表时间：
2008-09-19
影响因子：
4.8
通讯作者：
McDonough, Michael A.
中科院分区：
文献类型：
--
作者：
Hewitson, Kirsty S.;Holmes, Samantha L.;McDonough, Michael A.
铁结合残基的2-His-1-羧酸三联体存在于许多非血红素铁加氧酶中，包括Fe（II）和2-酮戊二酸（2 OG）依赖性双加氧酶。制备并分析了天冬酰胺酰羟化酶（HIF抑制因子（FIH））的铁结合Asp-201残基的三种变体（D201 A、D201 E和D201 G）。FIH-D201 A和FIH-D201 E不催化天冬酰胺酰羟基化，但在还原剂的存在下，与野生型FIH相比，它们显示出增强的2 OG周转。FIH-D201 A对2 OG的转换通过添加HIF-1 α（786-826）肽而显著刺激。与FIH-D201 A和D201 E一样，D201 G变体增强了2 OG周转，但出乎意料地催化了天冬酰胺酰羟基化。与Fe（II）/Zn（II）、2 OG和HIF-1 α（786-826/788-806）复合的FIH-D201 A和D201 G变体的晶体结构暗示金属结合仅需要两个基于FIH的残基（His-199和His-279）。结果表明，2 OG依赖的双加氧酶铁连接残基的变化作为一种手段的功能分配应谨慎对待。根据最近对非血红素铁和2 OG依赖性卤化酶的生物化学和结构分析，该结果具有机械感兴趣，该卤化酶类似于FIH-D201 A/G变体，因为它们仅使用两个His残基来连接铁。
A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1 alpha(786-826) peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1 alpha(786-826/788-806) implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron.