Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii
Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii
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DOI：
10.1042/bj20081161
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发表时间：
2009-03-01
影响因子：
4.1
通讯作者：
Nidetzky, Bernd
中科院分区：
文献类型：
--
作者：
Leitgeb, Stefan;Straganz, Grit D.;Nidetzky, Bernd
β-二酮裂解酶 Dke1 是来自约氏不动杆菌的同源四聚体 Fe2+ 依赖性双加氧酶。 Dke1 protoiner 采用了 cupin 蛋白超家族的单结构域 P 桶折叠特征，并具有单核非血红素 Fe2+ 中心，其中组氨酸残基三联体 His-62、His-64 和 His-104 与催化金属协调。为了提供 Dke1 的特殊金属位点与非血红素 Fe2+ 更广泛的 2-His-1-Glu/Asp 结合位点的结构-功能关系，我们用谷氨酸和天冬酰胺分别替换每个组氨酸残基，并比较 Fe2+ 和四种非天然催化非活性金属与野生型和突变酶的纯化脱辅基形式的结合。厌氧平衡微透析 (Fe2+) 和荧光滴定（Fe2+、Cu2+、Ni2+、Mn2+ 和 Zn2+）实验的结果表明，天然 Dke1 中存在两个广泛特异性的金属结合位点，它们以 5 μM（位点 I）和类似于 0.3 mM（位点 11）的解离常数 (K-d) 结合 Fe2+。除了用天冬酰胺取代 His-104 之外，每个突变都会破坏位点 I 处 Fe2+ 的结合，但不会破坏其他二价金属离子的结合，而位点 11 处的金属结合基本上不受影响。含有谷氨酸取代的 Dke1 突变体完全失活，并且不能通过外部 Fe2+ 进行功能补充。天冬酰胺取代 His-62 和 His-104 的突变体的 Fe2+ 催化中心活性 (k(cat)) 与野生型酶在与戊烷-2,4-二酮反应中的 k(cat) 值 8.5 s (1) 相比，分别降低了 140 倍和 220 倍。 H64N 突变体不具有催化能力，除非存在外部 Fe2+ (1 mM)，其引发的活性约为野生型的 1/1000。因此，Dkel 与 Fe2+ 的配位需要不带电的金属中心，并且需要三个组氨酸配体来组装全功能的催化位点。 Dke1 的氧化失活涉及酶结合的 Fe2+ 转化为 Fe2+，然后从金属中心释放。
beta-Diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1 protoiner adopts a single-domain P-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe2+ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp-binding site for non-haem Fe2+, we replaced each histicline residue individually with glutamate and asparagine and compared binding of Fe2+ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titration (Fe2+, Cu2+, Ni2+, Mn2+ and Zn2+) experiments revealed the presence of two broadly specific metal-binding sites in native Dke1 that bind Fe2+ with a dissociation constant (K-d) of 5 mu M (site I) and similar to 0.3 mM (site 11). Each mutation, except for the substitution of asparagine for His-104, disrupted binding of Fe2+, but not that of the other bivalent metal ions, at site I, while leaving metal binding at site 11 largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe2+. The Fe2+ catalytic centre activity (k(cat)) of mutants with asparagine Substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the k(cat) Value of 8.5 s (1) for the wild-type enzyme in the reaction with pentane-2,4-dione. The H64N mutant was not catalytically competent, except in the presence of external Fe2+ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore co-ordination of Fe2+ by Dkel requires an uncharged metallocentre, and three histidine ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke1 was shown to involve conversion of enzymic-bound Fe2+ into Fe2+, which is then released from the metal centre.