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首页> 外文期刊>Biochemistry >Identification of function and mechanistic insights of guanine deaminase from Nitrosomonas europaea: Role of the C-terminal loop in catalysis
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Identification of function and mechanistic insights of guanine deaminase from Nitrosomonas europaea: Role of the C-terminal loop in catalysis

机译：欧洲亚硝基鸟嘌呤鸟嘌呤脱氨酶的功能鉴定和机理见解：C末端环在催化中的作用

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摘要

NE0047 from Nitrosomonas europaea has been annotated as a zinc-dependent deaminase; however, the substrate specificity is unknown because of the low level of structural similarity and sequence identity compared to other family members. In this study, the function of NE0047 was established as a guanine deaminase (catalytic efficiency of 1.2 × 10~5 M~(-1) s~(-1)), exhibiting secondary activity towards ammeline. The structure of NE0047 in the presence of the substrate analogue 8-azaguanine was also determined to a resolution of 1.9 ?. NE0047 crystallized as a homodimer in an asymmetric unit. It was found that the extreme nine-amino acid C-terminal loop forms an active site flap; in one monomer, the flap is in the closed conformation and in the other in the open conformation with this loop region exposed to the solvent. Calorimetric data obtained using the full-length version of the enzyme fit to a sequential binding model, thus supporting a cooperative mode of ligand occupancy. In contrast, the mutant form of the enzyme (ΔC) with the deletion of the extreme nine amino acids follows an independent model of ligand occupancy. In addition, the ΔC mutant also does not exhibit any enzyme activity. Therefore, we propose that the progress of the reaction is communicated via changes in the conformation of the C-terminal flap and the closed form of the enzyme is the catalytically active form, while the open form allows for product release. The catalytic mechanism of deamination was also investigated, and we found that the mutagenesis of the highly conserved active site residues Glu79 and Glu143 resulted in a complete loss of activity and concluded that they facilitate the reaction by serving as proton shuttles.
机译：来自亚硝基亚硝胺的NE0047已被注释为锌依赖性脱氨酶。然而，由于与其他家族成员相比结构相似性和序列同一性较低，因此底物特异性未知。在这项研究中，NE0047的功能被确立为鸟嘌呤脱氨酶（催化效率为1.2×10〜5 M〜（-1）s〜（-1）），对氨甲啉具有次要活性。在底物类似物8-氮杂鸟嘌呤存在下，NE0047的结构也被确定为1.9Ω的分辨率。 NE0047在不对称单元中结晶为均二聚体。发现极端的九个氨基酸的C-末端环形成了活性位点皮瓣。在一个单体中，瓣处于闭合构型，而在另一个处于开放构型中，该环区暴露于溶剂。使用酶的全长版本获得的量热数据适合顺序结合模型，从而支持配体占据的协同模式。相反，具有九个极端氨基酸缺失的酶（ΔC）突变形式遵循配体占有率的独立模型。另外，ΔC突变体也不显示任何酶活性。因此，我们建议反应的进展是通过改变C末端襟翼的构象来传达的，酶的封闭形式是催化活性形式，而开放形式则允许产物释放。还研究了脱氨的催化机理，我们发现高度保守的活性位点残基Glu79和Glu143的诱变导致活性完全丧失，并得出结论，它们通过充当质子穿梭而促进了反应。

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