Direct n.m.r. evidence for substrate-induced conformational changes in a beta-lactamase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Beta-lactam antibiotics are crucial in treating bacterial infections.
• Beta-lactamases are enzymes that confer antibiotic resistance by hydrolyzing beta-lactam rings.
• Bacillus licheniformis beta-lactamase (BLA) is a key enzyme in understanding antibiotic resistance mechanisms.

Purpose of the Study:

• To investigate the interaction between cefoxitin, a beta-lactam antibiotic, and Bacillus licheniformis beta-lactamase.
• To characterize the acyl-enzyme intermediate formed during the enzymatic reaction.
• To explore the potential of structural studies for understanding enzyme-inhibitor interactions.

Main Methods:

• Kinetic analysis of the enzyme-cefoxitin system.
• Attempted crystallographic studies of the acyl-enzyme complex.
• Nuclear Magnetic Resonance (NMR) spectroscopy to observe the acyl-enzyme intermediate.

Main Results:

• Cefoxitin and similar antibiotics are poor substrates for Bacillus licheniformis beta-lactamase.
• Crystallographic studies failed to detect a stable crystalline acyl-enzyme complex.
• NMR spectroscopy directly observed stable acyl-enzyme complexes with cefoxitin and moxalactam, showing distinct spectral modifications indicative of enzyme structural changes.

Conclusions:

• The interaction between cefoxitin/moxalactam and BLA forms a stable acyl-enzyme intermediate.
• NMR is a valuable tool for studying enzyme-antibiotic interactions and structural dynamics, even when crystallography is challenging.
• The observed structural modifications provide insights into the mechanism of beta-lactamase inhibition and resistance.