Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug.
Biotechnol Lett
; 43(1): 203-211, 2021 Jan.
Article
in En
| MEDLINE
| ID: mdl-32851465
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.Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Bacillus subtilis
/
Bacterial Proteins
/
Nitroreductases
/
Directed Molecular Evolution
/
Nitroimidazoles
Language:
En
Journal:
Biotechnol Lett
Year:
2021
Document type:
Article
Affiliation country:
New Zealand