Characterization of Escherichia coli nitroreductase NfsB in the metabolism of nitrobenzodiazepines.
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.
Linwu
Shiuan-Woei
SW
Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan.
Syu
Cheng-Jie
CJ
Chen
Yu-Lian
YL
Wang
Andrew H-J
AH
Peng
Fu-Chuo
FC
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
D029968:Escherichia coli Proteins; D009574:Nitro Compounds; D001569:Benzodiazepines; D005445:Flunitrazepam; C116054:NfsB protein, E coli; D009601:Nitroreductases