Analysis of an enzymatic domain that is involved in specificity of phosphoryl donors, polyphosphate and ATP

无机聚磷酸盐（polyP）是一种由高能磷酸酐键连接的磷酸盐线性聚合物。珊瑚虫大量存在于火山凝析物中。因此，一些研究人员认为息肉可能是益生元生命的能量货币。我们发现磷小月牙体内存在一种严格依赖多肽的葡萄糖激酶。在许多生物体中，ATP被用来磷酸化葡萄糖，而polyP则被用于这种微生物。我们确定了该poly -葡糖激酶的初级结构，并发现ATP-和poly -葡糖激酶的活性位点残基都是保守的。然而，结构域IIB仅在atp -葡萄糖激酶中观察到。我们认为atp -葡萄糖激酶是由poly -葡萄糖激酶进化而来的。在这项研究中，我们希望揭示葡萄糖激酶获得结构域IIB并改变其磷酸化供体时的结构变化。首先，我们从重组大肠杆菌中纯化poly -glucokinase。纯化后的酶进一步浓缩并用于寻找结晶条件。最后，我们成功地进行了poly -glucokinase的结晶，并获得了两埃分辨率的x射线衍射图。我们还没有解决，但成功地预测了它的三维结构。我们预测poly -葡聚糖酶获得腺苷识别结构域并进化为atp -葡聚糖酶。

Inorganic polyphosphate (polyP) is a linear polymer of phosphate linked by high energy phosphoanhydride bonds. PolyP is found in abundance in volcanic condensates. Therefore, some investigators consider that polyP may be energy currency for prebiotic life. We found that a strictly polyP-dependent glucokinase in Microlunatus phosphovorus. ATP is used to phosphorylate glucose in many organisms, while polyP is used in this microorganism. We determined a primary structure of this polyP-glucokinase, and found that the active site residues are conserved in both of ATP- and polyP-glucokinase. However, a domain IIB is only observed in ATP-glucokinase. We considered that ATP-glucokinase is evolved from polyP-glucokinase. In this study, we wanted to reveal structural changes in glucokinase when it acquire the domain IIB and changes its phosphoryl donor. First we purified polyP-glucokinase from E.coli recombinant. The purified enzyme is further concentrated and used in order to find crystalization condition. Finally we succeeded in crystalization of polyP-glucokinase and obtaining X-ray diffraction pattern with two-angstrom resolution. We have not yet solved, but succeeded in predicting its three-dimensional structure. We predicted that polyP-glucokinase acquired adenosine recognizing domain and evolved to ATP-glucokinase.