3W0R

Crystal structure of a thermostable mutant of aminoglycoside phosphotransferase APH(4)-Ia (N202A), ternary complex with AMP-PNP and hygromycin B

Summary for 3W0R

• Entry DOI: 10.2210/pdb3w0r/pdb
• Related: 3W0M 3W0N 3W0O 3W0P 3W0Q 3W0S
• Descriptor: Hygromycin-B 4-O-kinase, PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER, HYGROMYCIN B VARIANT, ... (4 entities in total)
• Functional Keywords: phosphotransferase, transferase-antibiotic complex, transferase/antibiotic
• Biological source: Escherichia coli
• Total number of polymer chains: 1
• Total formula weight: 40067.48

Authors

Iino, D.,Takakura, Y.,Fukano, K.,Sasaki, Y.,Hoshino, T.,Ohsawa, K.,Nakamura, A.,Yajima, S. (deposition date: 2012-11-02, release date: 2013-08-07, Last modification date: 2024-03-20)

Primary citation

Iino, D.,Takakura, Y.,Fukano, K.,Sasaki, Y.,Hoshino, T.,Ohsawa, K.,Nakamura, A.,Yajima, S.
Crystal structures of the ternary complex of APH(4)-Ia/Hph with hygromycin B and an ATP analog using a thermostable mutant.
J.Struct.Biol., 183:76-85, 2013

PubMed 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.

PubMed: 23747390
DOI: 10.1016/j.jsb.2013.05.023

Experimental method

X-RAY DIFFRACTION (2.3 Å)