Nitroreductase catalyzed biotransformation of CL-20.
Previously, we reported that a salicylate 1-monooxygenase from Pseudomonas sp. ATCC 29352 biotransformed CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane) (C(6)H(6)N(12)O(12)) and produced a key metabolite with mol. wt. 346 Da corresponding to an empirical formula of C(6)H(6)N(10)O(8) which spontaneously decomposed in aqueous medium to produce N(2)O, NH(4)(+), and HCOOH [Appl. Environ. Microbiol. (2004)]. In the present study, we found that nitroreductase from Escherichia coli catalyzed a one-electron transfer to CL-20 to form a radical anion (CL-20(-)) which upon initial N-denitration also produced metabolite C(6)H(6)N(10)O(8). The latter was tentatively identified as 1,4,5,8-tetranitro-1,3a,4,4a,5,7a,8,8a-octahydro-diimidazo[4,5-b:4',5'-e]pyrazine [IUPAC] which decomposed spontaneously in water to produce glyoxal (OHCCHO) and formic acid (HCOOH). The rates of CL-20 biotransformation under anaerobic and aerobic conditions were 3.4+/-0.2 and 0.25+/-0.01 nmol min(-1)mg of protein(-1), respectively. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.8 nitrite ions, 3.3 molecules of nitrous oxide, 1.6 molecules of formic acid, 1.0 molecule of glyoxal, and 1.3 ammonium ions. Carbon and nitrogen products gave mass-balances of 60% and 81%, respectively. A comparative study between native-, deflavo-, and reconstituted-nitroreductase showed that FMN-site was possibly involved in the biotransformation of CL-20.
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2.
Bharat
B
Bhushan
Annamaria
A
Halasz
Jalal
J
Hawari
D001372:Aza Compounds / Q000737:chemistry*; D001665:Binding Sites; D001711:Biotransformation; D002384:Catalysis; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry; D006037:Glyoxal / Q000737:chemistry*; D006571:Heterocyclic Compounds / Q000737:chemistry*; D007700:Kinetics; D009601:Nitroreductases / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D011719:Pyrazines / Q000737:chemistry*
C482139:2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane; D001372:Aza Compounds; D029968:Escherichia coli Proteins; D005415:Flavins; D006571:Heterocyclic Compounds; D011719:Pyrazines; D006037:Glyoxal; C116053:NfsA protein, E coli; D009601:Nitroreductases; D010100:Oxygen