The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two l-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 l-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9–11) and a substrate affinity of ∼1.3 mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser–Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.

中文翻译：

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根瘤菌有两种L-天冬酰胺酶，序列同一性较低，但结构和催化中心相似

根瘤菌 ( Rhizobium etli)是豆科植物的固氮细菌共生体，其基因组编码两种L-天冬酰胺酶 ReAIV 和 ReAV，它们与已充分表征的 1 类（细菌型）和 2 类（植物型）酶没有相似性。据推测，ReAIV 和 ReAV 可能属于相同的结构类别 3，尽管它们的序列同一性水平较低。当诱导型热不稳定蛋白 ReAV 的晶体结构得到解决后，这一假设获得了更坚实的基础，因为 ReAV 的关键残基也存在于组成型热稳定 ReAIV 蛋白的序列中。ReAIV 的高分辨率晶体结构现在证实它是一种 3 类l-天冬酰胺酶，结构与 ReAV 相似，但有重要差异。最显着的差异涉及两种蛋白质的特殊水合模式、ReAIV 中三个内部空腔的存在以及锌结合位点的行为。ReAIV 具有较高的最适 pH 值 (9–11)，在 pH 9.0 时底物亲和力为 ∼1.3 m M。这些参数不适合 ReAIV 作为抗白血病药物的直接应用，尽管其热稳定性和缺乏谷氨酰胺酶活性将具有相当大的优势。这项工作中提出的 ReAIV 的五种晶体结构允许假设一种可能的酶促场景，其中在活性位点配位的锌离子是可有可无的元素。催化亲核试剂似乎是 Ser47，它是活性位点中两个 Ser-Lys 串联的一部分。这里介绍的 ReAIV 结构可以为未来的酶工程实验提供基础，以改善医学应用的动力学参数。