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Converting the E. coli Isochorismatase Nicotinamidase into γ-Lactamase
Xiaoyan Guo1,2, Licao Chang3, Haibo Jin1,2
1College of New Materials and Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing, People's Republic of China.
Microbiology Spectrum
|February 16, 2022
Summary
Nicotinamidase (Nic) was engineered into a (+) γ-lactamase by removing a loop, revealing the isochorismatase superfamily
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Nicotinamidase (Nic) from Escherichia coli, an isochorismatase superfamily member, shares active site similarities with known (+) γ-lactamases.
- This suggests Nic may possess latent (+) γ-lactamase activity.
Purpose of the Study:
- To investigate the latent (+) γ-lactamase potential within the isochorismatase superfamily.
- To engineer Nic into an active (+) γ-lactamase through structural modification.
Main Methods:
- Sequence alignment of five E. coli isochorismatase superfamily proteins to identify key structural features.
- Site-directed mutagenesis to delete a six-residue loop (112GENPLV117) in Nic.
- Characterization of the engineered protein's enzymatic activity.
Main Results:
- Deletion of the loop converted Nic into an active (+) γ-lactamase.
- The engineered enzyme exhibits a more compact binding pocket, stabilizing substrates and intermediates.
- Another latent (+) γ-lactamase within the superfamily was identified and activated.
Conclusions:
- The isochorismatase superfamily is a promising source for novel (+) γ-lactamases.
- Engineering substrate access tunnels, like the loop deletion in Nic, is effective for enzyme conversion and enhancing promiscuity.
- The observed activities suggest evolutionary pathways for γ-lactamase development within this superfamily.
