Unveiling the evolution routes of TEM-type extended-spectrum β-lactamases

The TEM-1 β-lactamase can only cleave penicillin and the first-generation cephalosporins but it has evolved to become active against second-, third- and fourth-generation drugs. Through sequence analysis of natural TEM variants and those created by mutagenesis experiments, we described two distinct evolution routes of TEM-1 that has generated over 220 enzyme variants. One began with the Gly²³⁸Ser alteration and the other originated with the Arg¹⁶⁴Ser substitution. Further acquisition of mutations in the background of each of these two first-step mutants led to stepwise alteration in enzyme structure and hence activity, eventually producing a wide range of enzyme variants whose substrate specificities cover cephalosporins of all generations. Dissemination of strains producing TEM-1 variants generated from these two evolution routes underlies the markedly increased prevalence of bacterial resistance to β-lactams in the past few decades. This study provides insights into the evolution of hydrolysing enzymes, in particular β-lactamases.