Leitgeb Stefan, Straganz Grit D, Nidetzky Bernd
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria.
Biochem J. 2009 Mar 1;418(2):403-11. doi: 10.1042/BJ20081161.
beta-diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1protomer adopts a single-domain beta-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 histidine 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 (Kd) of 5 microM (site I) and approximately 0.3 mM (site II). 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 II largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe2+.The Fe2+ catalytic centre activity (kcat) of mutants with asparagine substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the kcat 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 Dke1 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 enzyme-bound Fe2+ into Fe3+, which is then released from the metal centre.
β-二酮裂解酶Dke1是一种来自约翰逊不动杆菌的同源四聚体Fe2+依赖性双加氧酶。Dke1原体采用了蛋白质杯形超家族特有的单结构域β桶折叠结构，其特征是具有一个单核非血红素Fe2+中心，其中组氨酸残基His-62、His-64和His-104的三联体与催化金属配位。为了提供Dke1独特金属位点与更广泛的非血红素Fe2+的2-His-1-Glu/Asp结合位点相关的结构-功能关系，我们将每个组氨酸残基分别替换为谷氨酸和天冬酰胺，并比较了Fe2+和四种非天然催化无活性金属与野生型和突变型酶的纯化脱辅基形式的结合情况。厌氧平衡微透析（Fe2+）和荧光滴定（Fe2+、Cu2+、Ni2+、Mn2+和Zn2+）实验结果表明，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的解离常数（Kd）（位点I）和大约0.3 mM（位点II）结合Fe2+。除了用天冬酰胺取代His-104外，每个突变都破坏了位点I处Fe2+的结合，但不影响其他二价金属离子的结合，而位点II处的金属结合基本不受影响。携带谷氨酸替代的Dke1突变体完全无活性，且不能被外部Fe2+功能互补。与野生型酶在与戊烷-2,4-二酮反应中的8.5 s-1的kcat值相比，His-62和His-104被天冬酰胺替代的突变体的Fe2+催化中心活性（kcat）分别降低了140倍和220倍。H64N突变体没有催化活性，除非存在外部Fe2+（1 mM），此时其活性约为野生型活性的1/1000。因此，Dke1对Fe2+的配位需要一个不带电荷的金属中心，并且组装一个完全功能性的催化位点需要三个组氨酸配体。已证明Dke1的氧化失活涉及酶结合的Fe2+转化为Fe3+，然后从金属中心释放。
