<p>Five <i>Escherichia coli</i> proteins in the isochorismatase superfamily (EntB, RutB, Nic, YcaC, and YecD) were cloned and expressed. Among them, only RutB exhibited ( +) γ-lactamase activity. The primary structures of these five proteins were compared to those of a ( +) γ-lactamase (Mhpg) from <i>Microbacterium hydrocarbonoxydans</i>. Subsequently, the active site constellations (ASCs) of the proteins were superimposed. By imitating the ASCs of Mhpg and RutB, a single mutation converted YecD into an active ( +) γ-lactamase (YecD-G145C). A mutant with three mutations (YecD-G145C-W115E-V67I) engineered through combinatorial saturation mutagenesis was created. The catalytic efficiency (<i>k</i>_<i>cat</i><i>/K</i>_<i>m</i>) of this mutant was 31-fold higher than that of YecD-G145C. Furthermore, the specific production rate (SPR) of the triple mutant (106 ± 4&#xa0;mg/h·g dry cell weight, DCW) exceeded those of both RutB (89 ± 3&#xa0;mg/h·g DCW) and Mhpg (46 ± 1&#xa0;mg/h·g DCW), underscoring its superior catalytic robustness. The discovery of RutB and the latent ( +) γ-lactamase activity of YecD suggests that several members of the isochorismatase superfamily remain to be discovered, and members of this family could be used to identify novel ( +) γ-lactamases. Some of the members, such as YecD, could be engineered into robust catalysts.</p> Graphical abstract <p></p>