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.