title: An unusually cold active nitroreductase for prodrug activations.
abstract: A set of PCR primers based on the genome sequence were used to clone a gene encoding a hypothetical nitroreductases (named as Ssap-NtrB) from uropathogenic staphylococcus, Staphylococcus saprophyticus strain ATCC 15305, an oxygen insensitive flavoenzyme. Activity studies of the translation product revealed that the nitroreductase catalyses two electron reduction of a nitroaromatic drug of nitrofurazone (NFZ), cancer prodrugs of CB1954 and SN23862 at optimum temperature of 20 °C together with retaining its maximum activity considerably at 3 °C. The required electrons for such reduction could be supplied by either NADH or NADPH with a small preference for the latter. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a mostly soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined. The findings lead us to propose that Ssap-NtrB represents a novel nitro reductase with an unusual cold active property, which has not been described previously for prodrug activating enzymes of nitroreductases.
authors.affiliation: Gebze Institute of Technology, Department of Chemistry, 41400 Gebze-Kocaeli, Turkey. ayhancelik@gyte.edu.tr
authors.forename: Ayhan
authors.initials: A
authors.lastname: Çelik
authors.forename: Gülden
authors.initials: G
authors.lastname: Yetiş
mesh_terms: D000816:Aniline Mustard / Q000031:analogs & derivatives / Q000378:metabolism; D001388:Aziridines / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005486:Flavin Mononucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D009583:Nitrofurazone / Q000378:metabolism; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011355:Prodrugs / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D057790:Staphylococcus saprophyticus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D001388:Aziridines; D011355:Prodrugs; D011994:Recombinant Proteins; C076205:5-(N,N-bis(2-chloroethyl)amino)-2,4-dinitrobenzamide; C100099:tretazicar; D005486:Flavin Mononucleotide; D000816:Aniline Mustard; D009601:Nitroreductases; D009583:Nitrofurazone