Mutation of the conserved Asp-Asp pair impairs the structure, function, and inhibition of CTX-M Class A β-lactamase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Class A beta-lactamases are enzymes that hydrolyze beta-lactam antibiotics, conferring bacterial resistance.
• The Asp233-Asp246 catalytic dyad is a highly conserved motif in these enzymes.
• Understanding the precise function of this dyad is critical for developing new antibiotics.

Purpose of the Study:

• To characterize the functional role of the conserved Asp233-Asp246 pair in CTX-M-14 beta-lactamase.
• To investigate the impact of mutations within this pair on enzyme activity, substrate specificity, and inhibitor binding.

Main Methods:

• Site-directed mutagenesis was used to create the D233N mutant of CTX-M-14.
• Enzyme kinetics (kcat/Km) were measured for various substrates, including nitrocefin and cefotaxime.
• Inhibitor binding affinity was assessed.
• Structural analysis of the mutant enzyme was performed.
• Further mutagenesis of Thr216 was conducted.

Main Results:

• The D233N mutation significantly reduced CTX-M-14 activity in a substrate-dependent manner, with greater impact on bulky substrates like cefotaxime.
• Inhibitor binding was reduced 10-fold in the D233N mutant.
• Structural changes included movement of the 213-219 loop and loss of a key hydrogen bond (Thr216-Thr235).
• Restoration of the hydrogen bond was observed upon inhibitor binding.
• Mutagenesis of Thr216 confirmed its contribution to CTX-M activity.

Conclusions:

• The Asp233-Asp246 pair is essential for CTX-M hydrolysis, particularly for substrates with bulky side chains.
• Increased protein flexibility, influenced by residues like Thr216 and loop dynamics, may be a mechanism for evolving resistance to beta-lactam antibiotics.