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Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas
Takamitsu Furuyama1, Yoshikazu Ishii, Norimasa Ohya
1Department of Material and Biological Chemistry, Graduate School of Science and Engineering, Yamagata University, 1-4-12 Kojirakawa, Yamagata 990-8560, Japan.
Abstract:
Class B β-lactamases are known as metallo-β-lactamases (MBLs) and they hydrolyze most β-lactams, including carbapenems. IMP-18, an MBL cloned from Pseudomonas aeruginosa, was overexpressed, purified and crystallized by vapour diffusion for X-ray crystallographic analysis. Preliminary X-ray analysis showed that the crystal diffracted to 2.4 Å resolution and belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 120.77, c = 96.54 Å, α = β = γ = 90°, suggesting the presence of two molecules in the asymmetric unit.
Insights
This study focused on IMP-18, a metallo-β-lactamase (MBL) from Pseudomonas aeruginosa. Researchers determined its crystal structure to understand its function in hydrolyzing critical antibiotics.
Area of Science:
- Structural Biology
- Microbiology
- Biochemistry
Background:
- Class B β-lactamases, or metallo-β-lactamases (MBLs), are enzymes capable of hydrolyzing a broad spectrum of β-lactam antibiotics, including carbapenems.
- The emergence of MBLs poses a significant threat to infectious disease treatment due to their resistance to existing β-lactamase inhibitors.
- IMP-18 is a notable MBL identified in Pseudomonas aeruginosa, necessitating structural and functional characterization.
Purpose of the Study:
- To elucidate the three-dimensional structure of the IMP-18 metallo-β-lactamase.
- To provide insights into the catalytic mechanism and substrate specificity of IMP-18.
- To facilitate the development of novel inhibitors targeting MBLs.
Main Methods:
- Overexpression and purification of the IMP-18 enzyme from cloned Pseudomonas aeruginosa.
- Crystallization of purified IMP-18 using vapor diffusion techniques.
- X-ray crystallographic analysis of the IMP-18 crystals to determine the structure.
Main Results:
- The IMP-18 crystal diffracted X-rays to a resolution of 2.4 Å.
- The crystal belonged to the tetragonal space group P4(1)2(1)2.
- Unit-cell parameters were determined as a = b = 120.77 Å and c = 96.54 Å, indicating two molecules per asymmetric unit.
Conclusions:
- The determined crystal structure provides a high-resolution model of IMP-18.
- The structural data will be crucial for understanding the molecular basis of IMP-18 activity.
- This work lays the foundation for structure-based drug design against carbapenem-resistant bacteria.
