title: Crystal structures of the ternary complex of APH(4)-Ia/Hph with hygromycin B and an ATP analog using a thermostable mutant.
abstract: 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°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.
authors.affiliation: Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Iino
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Takakura
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Fukano
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Takayuki
authors.initials: T
authors.lastname: Hoshino
authors.forename: Kanju
authors.initials: K
authors.lastname: Ohsawa
authors.forename: Akira
authors.initials: A
authors.lastname: Nakamura
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Yajima
mesh_terms: D001665:Binding Sites; D003461:Crystallography; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006921:Hygromycin B / Q000737:chemistry*; D019868:Kanamycin Kinase / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D055550:Protein Stability; D013696:Temperature
chemical_list: D006921:Hygromycin B; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D019868:Kanamycin Kinase
keywords: Aminoglycoside antibiotics; Conformational change; Crystal structure; Kinase; Thermostability