Crystal structures of the ternary complex of APH(4)-Ia/Hph with hygromycin B and an ATP analog using a thermostable mutant
Crystal structures of the ternary complex of APH(4)-Ia/Hph with hygromycin B and an ATP analog using a thermostable mutant
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DOI：
10.1016/j.jsb.2013.05.023
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发表时间：
2013-07-01
影响因子：
3
通讯作者：
Yajima, Shunsuke
中科院分区：
文献类型：
--
作者：
Iino, Daisuke;Takakura, Yasuaki;Yajima, Shunsuke
氨基糖苷 4-磷酸转移酶-Ia (APH(4)-Ia)/潮霉素 B 磷酸转移酶 (Hph) 通过磷酸化使氨基糖苷类抗生素潮霉素 B (hygB) 失活。最近报道了 APH(4)-Ia 与 hygB 的二元复合物的晶体结构。为了表征酶对底物的识别，我们确定了不可水解的 ATP 类似物 AMP-PNP 和 hygB 与野生型、热稳定 Hph 突变体 Hph5 和 apo 突变体酶形式的三元复合物的晶体结构。三元复合物和apo结构之间的比较表明，Hph在AMP-PNP和hygB结合后经历结构域移动。这大约是 APH(9)-Ia 病例的一半。我们还确定了突变体的晶体结构，其中保守的、催化重要的残基 Asp198 和 Asn203 以及非保守的 Asn202 被转化为 Ala，揭示了 Asn202 对于催化的重要性。 Hph5 包含 5 个氨基酸取代，使其热稳定性改变 16 摄氏度；其结构表明，Hph5 的 4/5 突变位于疏水核心，似乎通过加强疏水相互作用来提高热稳定性。 (C) 2013 Elsevier Inc. 保留所有权利。
Aminoglycoside 4-phosphotransferase-Ia (APH(4)-Ia)/Hygromycin B phosphotransferase (Hph) inactivates the aminoglycoside antibiotic hygromycin B (hygB) via phosphorylation. The crystal structure of the binary complex of APH(4)-Ia with hygB was recently reported. To characterize substrate recognition by the enzyme, we determined the crystal structure of the ternary complex of non-hydrolyzable ATP analog AMP-PNP and hygB with wild-type, thermostable Hph mutant Hph5, and apo-mutant enzyme forms. The comparison between the ternary complex and apo structures revealed that Hph undergoes domain movement upon binding of AMP-PNP and hygB. This was about half amount of the case of APH(9)-Ia. We also determined the crystal structures of mutants in which the conserved, catalytically important residues Asp198 and Asn203, and the non-conserved Asn202, were converted to Ala, revealing the importance of Asn202 for catalysis. Hph5 contains five amino acid substitutions that alter its thermostability by 16 degrees C; its structure revealed that 4/5 mutations in Hph5 are located in the hydrophobic core and appear to increase thermostability by strengthening hydrophobic interactions. (C) 2013 Elsevier Inc. All rights reserved.