Exploring the coupling of dynamic residues in TEM-1 β-lactamase and their role in activity.

Abstract

The increase in the incidence of β-lactamase enzymes resistant to inhibitors has elevated the search for novel therapeutic strategies, particularly those that target amino acid residues other than the active site. In this respect, correlated amino acids that tend to evolve and have direct or indirect communication with the active site are considered potential candidates. Thus, this study identified residues that are dynamically coupled to the active site of TEM-1 β-lactamase by using the dynamic coupling index. These residues were found to cluster in three distinct structural regions: in a loop close to one face of the active site (D214, K215, and V216), at the binding interface with β-lactamase inhibitory protein close to a second face of the active site (E104, Y105, S106, E110, T114), and on a β-strand at the N-terminus, distant from the active site (R43, V44). Representative residues, K215, E104, E110, T114, and R43, were selected for mutational analysis to assess their roles in enzyme activity. Notably, the mutation of R43 to alanine led to a significant reduction in activity, with up to 70%. To assess the role of the positive charge at R43, the R43K and R43E point mutants were generated, both of which exhibited complete loss of enzymatic activity. Circular dichroism analyses, thermal melting experiments, and the low expression levels pointed out a loss of stability for R43 mutants. These findings highlight R43 as a promising new target for the development of alternative β-lactamase inhibitors.