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.

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.

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