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Updated: Mar 18, 2026
A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
Structural and Mutagenic Analysis of Metallo-β-Lactamase IMP-18
Takamitsu Furuyama1, Haruka Nonomura2, Yoshikazu Ishii3
1Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata, Japan.
Abstract:
IMP-type metallo-β-lactamases (MBLs) are exogenous zinc metalloenzymes that hydrolyze a broad range of β-lactams, including carbapenems. Here we report the crystal structure of IMP-18, an MBL cloned from Pseudomonas aeruginosa, at 2.0-Å resolution. The overall structure of IMP-18 resembles that of IMP-1, with an αβ/βα "folded sandwich" configuration, but the loop that covers the active site has a distinct conformation. The relationship between IMP-18's loop conformation and its kinetic properties was investigated by replacing the amino acid residues that can affect the loop conformation (Lys44, Thr50, and Ile69) in IMP-18 with those occupying the corresponding positions in the well-described enzyme IMP-1. The replacement of Thr50 with Pro considerably modified IMP-18's kinetic properties, specifically those pertaining to meropenem, with the kcat/Km value increased by an order of magnitude. The results indicate that this is a key residue that defines the kinetic properties of IMP-type β-lactamases.
Insights
Metallo-β-lactamases (MBLs) are enzymes that break down antibiotics. Researchers studied IMP-18, revealing a key residue, Thr50, that significantly impacts its antibiotic-degrading properties.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Metallo-β-lactamases (MBLs) are critical enzymes conferring resistance to broad-spectrum β-lactam antibiotics, including carbapenems.
- IMP-type MBLs are a significant class of these enzymes, posing a growing threat in clinical settings.
Purpose of the Study:
- To determine the crystal structure of IMP-18, an IMP-type metallo-β-lactamase.
- To investigate the structural basis for IMP-18's kinetic properties and identify key residues influencing its activity.
Main Methods:
- X-ray crystallography was employed to obtain the 2.0-Å resolution structure of IMP-18.
- Site-directed mutagenesis was used to replace specific amino acid residues in IMP-18 with those from IMP-1 to assess functional impact.
Main Results:
- The crystal structure of IMP-18 revealed a conserved αβ/βα folded sandwich fold, similar to IMP-1, but with a distinct active site loop conformation.
- Mutagenesis studies identified Threonine 50 (Thr50) as a critical residue; its replacement with Proline in IMP-18 significantly altered kinetic properties, particularly for meropenem hydrolysis (kcat/Km increased by an order of magnitude).
Conclusions:
- The distinct loop conformation in IMP-18 is influenced by specific amino acid residues.
- Thr50 is a key determinant of IMP-type β-lactamase kinetic properties, highlighting its importance in enzyme function and potential drug resistance mechanisms.
