Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug.

Abstract

OBJECTIVES:

To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954.

RESULTS:

Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitize E. coli host cells to at least 2.4-fold lower concentrations of SN33623 than the native enzyme. Two of these variants were able to be purified in a functional form, and in vitro assays revealed these were twofold and fourfold improved in kcat/KM with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in kcat/KM versus wild type YfkO in converting CB1954 to a genotoxic drug.

CONCLUSIONS:

The enhanced activation of the PET imaging probe SN33623 and CB1954 prodrug exhibited by the lead evolved variant of YfkO offers prospects for improved enzyme-prodrug therapy.

• Authors: Michelle H Rich, Abigail V Sharrock, Amir Ashoorzadeh, Adam V Patterson, Jeff B Smaill, David F Ackerley
• Journal: Biotechnology letters (Biotechnol Lett) Vol. 43 Issue 1 Pg. 203-211 (Jan 2021) ISSN: 1573-6776 [Electronic] Netherlands
• PMID: 32851465 (Publication Type: Journal Article)

Chemical References

• Antineoplastic Agents
• Aziridines
• Bacterial Proteins
• Nitroimidazoles
• SN33623
• tretazicar
• Nitroreductases

Topics

• Antineoplastic Agents (metabolism)
• Aziridines (metabolism)
• Bacillus subtilis (enzymology, genetics)
• Bacterial Proteins (genetics, metabolism)
• Directed Molecular Evolution (methods)
• Enzyme Therapy
• Nitroimidazoles (metabolism)
• Nitroreductases (genetics, metabolism)