# Converting Escherichia coli isochorismatase YecD into γ-lactamase

## TL;DR

Researchers converted a bacterial enzyme into a more efficient catalyst for a specific chemical reaction, suggesting new possibilities for enzyme engineering.

## Contribution

A single mutation transformed YecD into a γ-lactamase, and further mutations significantly improved its catalytic efficiency.

## Key findings

- YecD-G145C-W115E-V67I mutant showed 31-fold higher catalytic efficiency than YecD-G145C.
- The triple mutant's specific production rate exceeded RutB and Mhpg, indicating superior catalytic robustness.
- The latent γ-lactamase activity in YecD suggests undiscovered members of the isochorismatase superfamily with potential for engineering.

## Abstract

Five Escherichia coli 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 Microbacterium hydrocarbonoxydans. 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 (kcat/Km) of this mutant was 31-fold higher than that of YecD-G145C. Furthermore, the specific production rate (SPR) of the triple mutant (106 ± 4 mg/h·g dry cell weight, DCW) exceeded those of both RutB (89 ± 3 mg/h·g DCW) and Mhpg (46 ± 1 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.

The online version contains supplementary material available at 10.1186/s40643-025-00960-y.