"Mind the Gap": Raman Evidence for Rapid Inactivation of CTX-M-9 β-Lactamase Using Mechanism-Based Inhibitors that...

Area of Science:

• Biochemistry
• Microbiology
• Structural Biology

Background:

• CTX-M β-lactamases are a rapidly expanding family of extended-spectrum β-lactamases (ESBLs) in *Escherichia coli*, conferring resistance to β-lactam antibiotics.
• Despite belonging to class A, CTX-M enzymes exhibit low sequence identity to other class A β-lactamases and possess high hydrolytic activity against oxyimino-cephalosporins, even with smaller active sites.
• While generally inhibited by clinical agents like clavulanic acid, sulbactam, and tazobactam, emerging inhibitor resistance necessitates a deeper mechanistic understanding.

Purpose of the Study:

• To elucidate the mechanistic details of CTX-M-9 inactivation pathways using Raman microscopy.
• To investigate the role of specific residues, such as Ser130, in the inactivation mechanism.
• To provide insights for the development of novel inhibitors to combat antibiotic resistance.

Main Methods:

• Raman microscopy was employed to study the inactivation reaction of CTX-M-9 with clavulanic acid, sulbactam, and tazobactam.
• Reactions were characterized in both CTX-M-9 single crystals and solution.
• The study included an analysis of a CTX-M-9 S130G variant to assess the contribution of Ser130.

Main Results:

• A unique cross-linked species, likely involving Ser70 and Ser130, was observed during CTX-M-9 inactivation.
• Subsequent hydrolysis led to the formation of an acrylate species linked to Ser130, observed in solution within 25 ms.
• The CTX-M-9 S130G variant reacted with inhibitors but lacked the characteristic spectroscopic signature of the Ser130-linked species, supporting its role.

Conclusions:

• The study reveals a novel inactivation mechanism for CTX-M-9 involving a bridging cross-linked species.
• Understanding this mechanism, particularly the role of Ser130, is crucial for designing new inhibitors against resistant strains.
• This knowledge can inform strategies to overcome emerging inhibitor resistance in clinically significant ESBL-producing bacteria.