Structural Insight into Substrate Selectivity of Erwinia chrysanthemi L-asparaginase.
Structural Insight into Substrate Selectivity of Erwinia chrysanthemi L-asparaginase.
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DOI：
10.1021/acs.biochem.5b01351
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发表时间：
2016-03-01
期刊：
影响因子：
2.9
通讯作者：
Lavie A
中科院分区：
文献类型：
--
作者：
Nguyen HA;Su Y;Lavie A
细菌来源的 L-天冬酰胺酶是急性淋巴细胞白血病治疗的支柱。这些酶药物的作用机制与其消耗血液中氨基酸 L-天冬酰胺的能力有关。然而，细菌L-天冬酰胺酶的临床使用因其双重L-天冬酰胺酶和L-谷氨酰胺酶活性而变得复杂。后者尽管仅占总体活性的 10%，但也是造成所观察到的毒副作用的部分原因。因此，缺乏 L-谷氨酰胺酶活性的 L-天冬酰胺酶具有作为更安全药物的潜力。了解 L-天冬酰胺酶底物特异性的关键决定因素是开发具有降低毒性的酶变体的先决条件。在这里，我们展示了欧文氏菌菊-天冬酰胺酶与 L-天冬氨酸和 L-谷氨酸复合物的晶体结构。这些结构揭示了两种酶构象——开放和闭合——分别对应于非活性和活性状态。配体的结合诱导催化 Thr15 定位到其活性构象，这反过来又允许柔性 N 端环的排序和闭合。值得注意的是，L-天冬氨酸在诱导 Thr15 的主动定位方面比 L-谷氨酸更有效。讨论了解释该酶对 L-天冬酰胺而非 L-谷氨酰胺的偏好的结构元素，并为未来开发更具体的 L-天冬酰胺酶提供指导。
l-Asparaginases of bacterial origin are a mainstay of acute lymphoblastic leukemia treatment. The mechanism of action of these enzyme drugs is associated with their capacity to deplete the amino acid l-asparagine from the blood. However, clinical use of bacterial l-asparaginases is complicated by their dual l-asparaginase and l-glutaminase activities. The latter, even though representing only ∼10% of the overall activity, is partially responsible for the observed toxic side effects. Hence, l-asparaginases devoid of l-glutaminase activity hold potential as safer drugs. Understanding the key determinants of l-asparaginase substrate specificity is a prerequisite step toward the development of enzyme variants with reduced toxicity. Here we present crystal structures of the Erwinia chrysanthemil-asparaginase in complex with l-aspartic acid and with l-glutamic acid. These structures reveal two enzyme conformations—open and closed—corresponding to the inactive and active states, respectively. The binding of ligands induces the positioning of the catalytic Thr15 into its active conformation, which in turn allows for the ordering and closure of the flexible N-terminal loop. Notably, l-aspartic acid is more efficient than l-glutamic acid in inducing the active positioning of Thr15. Structural elements explaining the preference of the enzyme for l-asparagine over l-glutamine are discussed with guidance to the future development of more specific l-asparaginases.