A surface loop modulates activity of the Bacillus class D β-lactamases

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• Beta-lactamases are key to bacterial resistance against beta-lactam antibiotics.
• Class D beta-lactamases were historically found in Gram-negative bacteria, but recently discovered in Gram-positive species like Bacillus.
• Some Bacillus species produce two distinct class D beta-lactamases with differing catalytic activities.

Purpose of the Study:

• Investigate the kinetic and structural properties of class D beta-lactamases from Bacillus species.
• Determine the molecular basis for the differential activity observed between highly active and basal activity class D beta-lactamases.
• Elucidate the role of specific surface loop residues in modulating enzyme activity.

Main Methods:

• Amino acid sequence analysis of active (BPU-1) and inactive (BSU-2, BAT-2) Bacillus class D beta-lactamases.
• Structural and molecular docking studies to assess substrate binding.
• Site-directed mutagenesis to delete specific residues from surface loops of BSU-2 and BAT-2.

Main Results:

• Sequence analysis identified three additional amino acid residues in a surface loop of inactive enzymes.
• Structural and docking studies revealed that this elongated loop sterically hinders substrate binding.
• Deletion of these three residues in BSU-2 and BAT-2 significantly increased their catalytic activity.

Conclusions:

• A specific surface loop containing three extra amino acids is responsible for the reduced catalytic activity of certain Bacillus class D beta-lactamases.
• This loop imposes steric constraints that limit substrate access and binding.
• Modulation of this surface loop is a critical factor in regulating the activity of Bacillus class D beta-lactamases.