title: Identification of novel nitroreductases from Bacillus cereus and their interaction with the CB1954 prodrug.
abstract: Directed enzyme prodrug therapy is a form of cancer chemotherapy in which bacterial prodrug-activating enzymes, or their encoding genes, are directed to the tumour before administration of a prodrug. The prodrug can then be activated into a toxic drug at the tumour site, reducing off-target effects. The bacterial nitroreductases are a class of enzymes used in this therapeutic approach and although very promising, the low turnover rate of prodrug by the most studied nitroreductase enzyme, NfnB from Escherichia coli (NfnB_Ec), is a major limit to this technology. There is a continual search for enzymes with greater efficiency, and as part of the search for more efficient bacterial nitroreductase enzymes, two novel enzymes from Bacillus cereus (strain ATCC 14579) have been identified and shown to reduce the CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) prodrug to its respective 2'-and 4'-hydroxylamine products. Both enzymes shared features characteristic of the nitro-FMN-reductase superfamily including non-covalently associated FMN, requirement for the NAD(P)H cofactor, homodimeric, could be inhibited by Dicoumarol (3,3'-methylenebis(4-hydroxy-2H-chromen-2-one)), and displayed ping pong bi bi kinetics. Based on the biochemical characteristics and nucleotide alignment with other nitroreductase enzymes, one enzyme was named YdgI_Bc and the other YfkO_Bc. Both B. cereus enzymes had greater turnover for the CB1954 prodrug compared with NfnB_Ec, and in the presence of added NADPH cofactor, YfkO_Bc had superior cell killing ability, and produced mainly the 4'-hydroxylamine product at low prodrug concentration. The YfkO_Bc was identified as a promising candidate for future enzyme prodrug therapy.
authors.affiliation: School of Chemistry, Bangor University, Bangor, Gwynedd, LL57 2DG Wales, UK.
authors.forename: Vanessa V
authors.initials: VV
authors.lastname: Gwenin
authors.affiliation: School of Chemistry, Bangor University, Bangor, Gwynedd, LL57 2DG Wales, UK.
authors.forename: Paramasivan
authors.initials: P
authors.lastname: Poornima
authors.affiliation: School of Chemistry, Bangor University, Bangor, Gwynedd, LL57 2DG Wales, UK.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Halliwell
authors.affiliation: School of Chemistry, Bangor University, Bangor, Gwynedd, LL57 2DG Wales, UK.
authors.forename: Patrick
authors.initials: P
authors.lastname: Ball
authors.affiliation: School of Chemistry, Bangor University, Bangor, Gwynedd, LL57 2DG Wales, UK.
authors.forename: George
authors.initials: G
authors.lastname: Robinson
authors.affiliation: School of Chemistry, Bangor University, Bangor, Gwynedd, LL57 2DG Wales, UK. Electronic address: c.d.gwenin@bangor.ac.uk.
authors.forename: Chris D
authors.initials: CD
authors.lastname: Gwenin
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism*; D001388:Aziridines / Q000378:metabolism*; D001409:Bacillus cereus / Q000201:enzymology*; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009601:Nitroreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011355:Prodrugs / Q000378:metabolism*; D011485:Protein Binding; D017421:Sequence Analysis; D013696:Temperature
chemical_list: D000970:Antineoplastic Agents; D001388:Aziridines; D011355:Prodrugs; C100099:tretazicar; D009601:Nitroreductases
keywords: Bacillus cereus; CB1954; Nitroreductase; Prodrug therapy