Converting the E. coli Isochorismatase Nicotinamidase into γ-Lactamase

Nicotinamidase (Nic) (E.C.3.5.1.19) is a representative protein of the isochorismatase superfamily from Escherichia coli. Despite showing no (+) γ-lactamase activity, its active site constellations (ASCs) are very similar to those of two other known (+) γ-lactamases (Mhpg and RutB), indicating that it could be a latent (+) γ-lactamase. In this study, the primary sequences of the five representative proteins of the isochorismatase superfamily from E. coli were aligned, and a "lid"-like unit of a six-residue loop (112GENPLV117) was established. The Nic protein was converted to a (+) γ-lactamase by eliminating the loop. A conversion mechanism was proposed in which a more compact binding pocket is formed after lid deletion. In addition, the "shrunk" binding pocket stabilized the small substrate and the catalysis intermediate, which triggered catalysis. Moreover, we identified another latent (+) γ-lactamase in the E. coli isochorismatase superfamily and successfully converted it into an active (+) γ-lactamase. In summary, the isochorismatase superfamily is potentially a good candidate for obtaining novel (+) γ-lactamases. γ-Lactamases are important enzymatic catalysts in preparing optically pure γ-lactam enantiomers, which are high-value chiral intermediates. Different studies have presumed that the isochorismatase superfamily is a candidate to obtain novel (+) γ-lactamases. By engineering its substrate entrance tunnel, Nic, a representative protein of the isochorismatase superfamily, is converted to a (+) γ-lactamase. Tunnel engineering has proven effective in enhancing enzyme promiscuity. Therefore, the latent or active γ-lactamase activities of the isochorismatase superfamily members indicate their evolutionary path positions.