Converting Escherichia coli isochorismatase YecD into γ-lactamase.

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 (k_cat/K_m) of this mutant was 31-fold higher than that of YecD-G145C. Furthermore, the specific produ

SUBMITTER: Guo X 

PROVIDER: S-EPMC12553813 | biostudies-literature | 2025 Oct

REPOSITORIES: biostudies-literature