Mutual influence of secondary and key drug‐resistance mutations on catalytic properties and thermal stability of TEM‐type β‐lactamases

Abstract

Highly mutable β‐lactamases are responsible for the ability of Gram‐negative bacteria to resist β‐lactam antibiotics. Using site‐directed mutagenesis technique, we have produced in vitro a number of recombinant analogs of naturally occurring TEM‐type β‐lactamases, bearing the secondary substitution Q39K and key mutations related to the extended‐spectrum (E104K, R164S) and inhibitor‐resistant (M69V) β‐lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of β‐lactamases. However, in combination with the key mutations, this substitution resulted in decreased KM values towards hydrolysis of a chromogenic substrate, CENTA. The ability of enzymes to restore catalytic activity after exposure to elevated temperature has been examined. All double and triple mutants of β‐lactamase TEM‐1 bearing the Q39K substitution showed lower thermal stability compared with the enzyme with Q39 intact. A sharp decrease in the stability was observed when Q39K was combined with E104K and M69V. The key R164S substitution demonstrated unusual ability to resist thermal inactivation. Computer analysis of the structure and molecular dynamics of β‐lactamase TEM‐1 revealed a network of hydrogen bonds from the residues Q39 and K32, related to the N‐terminal α‐helix, towards the residues R244 and G236, located in the vicinity of the enzyme's catalytic site. Replacement of Q39 by lysine in combination with the key drug resistance mutations may be responsible for loss of protein thermal stability and elevated mobility of its secondary structure elements. This effect on the activity of β‐lactamases can be used as a new potential target for inhibiting the enzyme.

Bacterial Enzymes and Antibiotic Resistance

“…These mutant forms are known as extended-spectrum β-lactamases (ESBLs). Certain mutations in amino acid residues located at a distance from the active site are compensating and may have multidirectional effects on stability [53, 54].…”

β-Lactamase-Mediated Fragmentation: Historical Perspectives and Recent Advances in Diagnostics, Imaging, and Antibacterial Design

“…However, the more modest color change is difficult to observe unaided, necessitating the use of specialized equipment, such as UV–vis spectrometers, that may be less practical for rapid, point-of-care diagnostics . Thus, while these novel probes represented a significant step forward from the original proof of concept (nitrocefin) and are widely used today, limitations remain regarding their utility for detection and diagnosis of resistant organisms in the clinic. − …”