Background: Glucose-6-phosphate isomerase (G6PI) catalyses the second step in glycolysis in the reversible interconversion of an aldohexose glucose 6-phosphate, a six membered ring moiety to a ketohexose, fructose 6-phosphate five membered ring moiety. This enzyme is of utmost importance due to its multifunctional role like neuroleukin, autocrine motility factor, etc. in various species. G6PI from Pseudomonas aeruginosa is less explored for its moonlighting properties. These properties can be predicted by studying the active site conservation of residues and their interaction with the specific ligand.

Methods: Here, we study the G6PI in a self-inducible construct in bacterial expression system with its purification using Ni-NTA chromatography. The secondary structure of pure G6PI is estimated using circular dichroism to further predict the proper folding form of the protein. The bioactivity of the purified enzyme is quantified using phosphoglucose isomerase colorimetric kit with a value of 12.5 mU/mL. Differential scanning fluorimetry and isothermal titration calorimetry were employed to monitor the interaction of G6PI with its competitive inhibitor, erythrose 4-phosphate and calculated the Tm, Kd and IC50 values. Further, the homology model for the protein was prepared to study the interaction with the erythrose 4-phosphate. MD simulation of the complex was performed at 100 ns to identify the binding interactions.

Results: We identified hydrogen bonds and water bridges dominating the interactions in the active site holding the protein and ligand with strong affinity.

Conclusion: G6PI was successfully crystallized and data has been collected at 6Å. We are focused on improving the crystal quality for obtaining higher resolution data.

背景：6-磷酸葡萄糖异构酶（G6PI）催化糖酵解中的第二步，即6-己糖醛糖葡萄糖（6-元环部分）可逆转化为酮己糖，果糖6-磷酸5元环部分。由于该酶在多种物种中具有多种功能，例如神经白蛋白，自分泌运动因子等，因此具有极为重要的意义。铜绿假单胞菌（Pseudomonas aeruginosa）的G6PI具有月光特性，因此鲜为人知。这些性质可以通过研究残基的活性位点保守性及其与特定配体的相互作用来预测。

方法：在这里，我们研究了细菌表达系统中自诱导构建体中的G6PI，并使用Ni-NTA色谱法对其进行了纯化。使用圆二色性估计纯G6PI的二级结构，以进一步预测蛋白质的正确折叠形式。使用磷酸葡萄糖异构酶比色试剂盒定量的纯化酶的生物活性为12.5 mU / mL。采用差示扫描荧光法和等温滴定热法监测G6PI及其竞争性抑制剂4-磷酸赤藓糖的相互作用，并计算Tm，Kd和IC50值。此外，制备蛋白质的同源性模型以研究与4-磷酸赤藓糖的相互作用。在100 ns进行复合物的MD模拟，以鉴定结合相互作用。

结果：我们确定了氢键和水桥主导着蛋白质和配体具有强亲和力的活性位点之间的相互作用。

结论：G6PI已成功结晶，并在6Å处收集了数据。我们专注于提高晶体质量以获得更高分辨率的数据。