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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• 作者：Daisuke Iino ; Yasuaki Takakura ; Kazuhiro Fukano ; Yasuyuki Sasaki ; Takayuki Hoshino ; Kanju Ohsawa ; Akira Nakamura ; Shunsuke Yajima
• 关键词：Aminoglycoside antibiotics ; Conformational change ; Crystal structure ; Kinase ; Thermostability
• 刊名：Journal of Structural Biology
• 出版年：2013
• 出版时间：July, 2013
• 年：2013
• 卷：183
• 期：1
• 页码：76-85
• 全文大小：2655 K

文摘

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 ¡ãC; its structure revealed that 4/5 mutations in Hph5 are located in the hydrophobic core and appear to increase thermostability by strengthening hydrophobic interactions.