Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn²⁺ is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn²⁺, which was established by measuring the activation constants (K_act) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity.

中文翻译：

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金属依赖的木糖异构酶从Piromyces sp。E2通过活性分析和蛋白质晶体学研究

来自Piromyces sp。的木糖异构酶。E2（PirXI）可用于使酿酒酵母具有将木糖发酵为乙醇的能力。尽管该酶的金属含量，催化参数和表达水平对于快速利用木糖至关重要，但尚未描述该酶的生化特性和结构。在大肠杆菌中高水平表达后，我们已经分离出了该酶，分析了其催化特性对金属的依赖性，并在存在不同金属，底物和底物类似物的情况下确定了12种晶体结构。活性测定表明，各种二价金属可以激活PirXI进行木糖异构化。在这些金属中，Mn ²⁺是最有利于催化活性的。此外，该酶显示出对Mn ²⁺的最高亲和力，这是通过测量不同金属的激活常数（K _act）确定的。对纯化酶的金属分析表明，该酶在体内与金属混合物结合，金属混合物由金属的可利用性和亲和力决定，表明天然金属成分可影响活性。晶体结构显示存在一个与其他木糖异构酶相似的活性位点，带有一个d-木糖结合位点，含有两个色氨酸和一个催化组氨酸，以及两个金属结合位点，它们是由保守的天冬氨酸和谷氨酸的羧酸盐基团形成的。对于不同的金属，金属配位残基的结合位置和构象略有不同，据推测这有助于观察到的异构酶活性对金属的依赖性。