ReAV, the inducible Class-3 L-asparaginase from the nitrogen-fixing symbiotic bacterium Rhizobium etli, is an interesting candidate for optimizing its enzymatic potential for antileukemic applications. Since it has no structural similarity to known enzymes with this activity, it may offer completely new ways of approach. Also, as an unrelated protein, it would evade the immunological response elicited by other asparaginases. The crystal structure of ReAV revealed a uniquely assembled protein homodimer with a highly specific C135/K138/C189 zinc binding site in each subunit. It was also shown before that the Zn2+ cation at low and optimal concentration boosts the ReAV activity and improves substrate specificity, which indicates its role in substrate recognition. However, the detailed catalytic mechanism of ReAV is still unknown. In this work, we have applied site-directed mutagenesis coupled with enzymatic assays and X-ray structural analysis to elucidate the role of the residues in the zinc coordination sphere in catalysis. Almost all of the seven ReAV muteins created in this campaign lost the ability to hydrolyze L-asparagine, confirming our predictions about the significance of the selected residues in substrate hydrolysis. We were able to crystallize five of the ReAV mutants and solve their crystal structures, revealing some intriguing changes in the active site area as a result of the mutations. With alanine substitutions of Cys135 or Cys189, the zinc coordination site fell apart and the mutants were unable to bind the Zn2+ cation. Moreover, the absence of Lys138 induced atomic shifts and conformational changes of the neighboring residues from two active-site Ser-Lys tandems. Ser48 from one of the tandems, which is hypothesized to be the catalytic nucleophile, usually changes its hydration pattern in response to the mutations. Taken together, the results provide many useful clues about the catalytic mechanism of the enzyme, allowing one to cautiously postulate a possible enzymatic scenario.

中文翻译：

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通过诱变探测 3 类 L-天冬酰胺酶的活性位点。 I. 修补 ReAV 的锌配位位点

ReAV 是来自固氮共生细菌 etli 的诱导型 3 类 L-天冬酰胺酶，是优化其抗白血病应用酶潜力的有趣候选者。由于它与具有这种活性的已知酶没有结构相似性，因此它可能提供全新的方法。此外，作为一种不相关的蛋白质，它会逃避其他天冬酰胺酶引起的免疫反应。 ReAV 的晶体结构揭示了一种独特组装的蛋白质同二聚体，每个亚基中都有高度特异性的 C135/K138/C189 锌结合位点。之前还表明，低浓度和最佳浓度的 Zn2+ 阳离子可增强 ReAV 活性并提高底物特异性，这表明其在底物识别中的作用。然而，ReAV的详细催化机制仍不清楚。在这项工作中，我们应用定点诱变结合酶测定和 X 射线结构分析来阐明锌配位层中残基在催化中的作用。在此活动中创建的几乎所有 7 个 ReAV 突变蛋白都失去了水解 L-天冬酰胺的能力，证实了我们对所选残基在底物水解中的重要性的预测。我们能够结晶出五个 ReAV 突变体并解析它们的晶体结构，揭示了突变导致的活性位点区域的一些有趣的变化。随着 Cys135 或 Cys189 的丙氨酸取代，锌配位点崩溃，突变体无法结合 Zn2+ 阳离子。此外，Lys138 的缺失会引起两个活性位点 Ser-Lys 串联的相邻残基的原子位移和构象变化。来自其中一个串联体的 Ser48，被假设为催化亲核试剂，通常会响应突变而改变其水合模式。总而言之，这些结果提供了有关酶催化机制的许多有用线索，使人们能够谨慎地假设一种可能的酶促情况。