title: Initial reaction(s) in biotransformation of CL-20 is catalyzed by salicylate 1-monooxygenase from Pseudomonas sp. strain ATCC 29352.
abstract: CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C(6)H(6)N(12)O(12)), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp. strain FA1 and Agrobacterium sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 +/- 0.011 and 0.043 +/- 0.003 nmol min(-1) mg of protein(-1) under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M - H](-) at 345 Da, corresponding to an empirical formula of C(6)H(6)N(10)O(8), produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M - H](-) at 381 Da corresponding to an empirical formula of C(6)H(10)N(10)O(10). The latter was a hydrated product of the metabolite C(6)H(6)N(10)O(8) with addition of two H(2)O molecules, as confirmed by tests using (18)O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical.
authors.affiliation: Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada.
authors.forename: Bharat
authors.initials: B
authors.lastname: Bhushan
authors.forename: Annamaria
authors.initials: A
authors.lastname: Halasz
authors.forename: Jim C
authors.initials: JC
authors.lastname: Spain
authors.forename: Jalal
authors.initials: J
authors.lastname: Hawari
mesh_terms: D001372:Aza Compounds / Q000378:metabolism*; D001673:Biodegradation, Environmental; D001711:Biotransformation; D002384:Catalysis; D006571:Heterocyclic Compounds / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000502:physiology*; D011549:Pseudomonas / Q000378:metabolism*
chemical_list: C482139:2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane; D001372:Aza Compounds; D006571:Heterocyclic Compounds; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase