title: Characterization of Escherichia coli nitroreductase NfsB in the metabolism of nitrobenzodiazepines.
abstract: Nitrobenzodiazepine (NBDZ) is a sedative-hypnotic drug used in the treatment of anxiety and sleep problems. Overdose of NBDZ may cause severe neurological effects, especially for people in drug abuse or addiction. In the present study, we investigated NBDZ nitroreduction in rat enteric contents and characterized the role of enterobacterial nitroreductase in the reductive pathway. Nitroreduction of flunitrazepam (FZ) was studied in the microsomal membrane fractions of rat liver, jejunum and jejunal microflora using HPLC analysis. In the jejunal microflora, FZ was demonstrated to be significantly reduced to its amino derivative under anaerobic condition. Escherichia coli type I nitroreductase NfsB (EC 1.5.1.34) was found in rat jejunal microflora via PCR technique and Western blotting. The participation of NfsB in FZ nitroreduction was demonstrated from inhibition studies. Kinetic study of the purified recombinant NfsB indicated that nitroreduction of FZ, nitrazepam (NZ) and clonazepam (CZ) are mediated by NfsB, where CZ shows lower k(cat)/K(M) ratio than that of the other two. Finally, two other nitroreductases E. cloacae NR (EC 1.6.99.7) and S. typhimurium Cnr were also found to be responsible for FZ nitroreduction. These results provide that the reduction of NBDZ in normal flora is catalyzed by type I nitroreductase NfsB.
authors.affiliation: Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Shiuan-Woei
authors.initials: SW
authors.lastname: Linwu
authors.forename: Cheng-Jie
authors.initials: CJ
authors.lastname: Syu
authors.forename: Yu-Lian
authors.initials: YL
authors.lastname: Chen
authors.forename: Andrew H-J
authors.initials: AH
authors.lastname: Wang
authors.forename: Fu-Chuo
authors.initials: FC
authors.lastname: Peng
mesh_terms: D000818:Animals; D001569:Benzodiazepines / Q000737:chemistry / Q000378:metabolism*; D001711:Biotransformation; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005445:Flunitrazepam / Q000737:chemistry / Q000378:metabolism; D007425:Intracellular Membranes / Q000382:microbiology; D007583:Jejunum / Q000382:microbiology; D008297:Male; D008861:Microsomes / Q000382:microbiology; D008862:Microsomes, Liver / Q000382:microbiology; D009574:Nitro Compounds / Q000737:chemistry / Q000378:metabolism*; D009601:Nitroreductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D012486:Salmonella typhimurium / Q000201:enzymology
chemical_list: D029968:Escherichia coli Proteins; D009574:Nitro Compounds; D001569:Benzodiazepines; D005445:Flunitrazepam; C116054:NfsB protein, E coli; D009601:Nitroreductases