Radical catalysis in Fe/S cluster dependent dehydratases

典型的脱水酶催化碳氧键的裂解，通过a，b消除水生成不饱和产物。以质子形式脱除的a-氢(pKa~8)被相邻的吸电子基团活化，羟基从b位离开。相比之下，几种厌氧细菌使用氧敏性2-或4-羟基酰辅酶A脱水酶，这种酶使用自由基将未激活的b质子的pk降低高达26个单位。这些非典型脱水酶是2-羟基酰辅酶A脱水酶和4-羟基丁酰辅酶A脱水酶。前者由两部分组成：(A)产生自由基的依赖Fe/S的ATPase组分(激活剂)和催化实际脱水的依赖Fe/S的脱水酶。最近的光谱研究表明，底物上生成了一个催化活性的酮基阴离子来促进脱水。研究建议侧重于阐明(I)的结构基础。为激活剂建议的核苷酸和氧化还原依赖的变化，(Ii)这两个组分之间的络合物形成导致脱水酶组分和(III.)脱水酶组分的不同寻常的活性中心金属簇。(四)此外，我们希望通过研究底物和产物复合体来扩展我们对4-羟丁酰辅酶A脱水酶机理的结构研究。

Typical dehydratases catalyze the cleavage of a carbon-oxygen bond yielding an unsaturated product via a,b-elimination of water. The a-hydrogen (pKa ~ 8) to be removed as proton is activated by an adjacent electron-withdrawing group, and the hydroxyl group leaves from the b-position. In contrast, several anaerobic bacteria employ oxygensensitive 2- or 4-hydroxyacyl-CoA dehydratases that use radicals to lower the pK of the unactivated b-protons by up to 26 units. These atypical dehydratases are the class of 2- hydroxyacyl-CoA dehydratases and the 4-hydroxybutyryl-CoA dehydratase. The former consist of two components: (a) a Fe/S-dependent ATPase component (activator) to generate the radical and (b) a Fe/S-dependent dehydratase catalyzing the actual dehydration. Recent spectroscopic investigations showed that a catalytically competent ketyl-radical anion is generated on the substrate to facilitate the dehydration. The research proposal focuses on elucidating the structural bases of (I.) nucleotide- and redox-dependent changes proposed for the activator, (II.) complex formation between the two components leading to radical generation on the dehydratase component and (III.) the unusual active site metal cluster of the dehydratase component. (IV.) Additionally, we want to extent our structural studies on the mechanism of 4-hydroxybutyryl-CoA dehydratases by studying substrate- and product-complexes.