Initial Reaction(s) in Biotransformation of CL-20 Is Catalyzed by Salicylate 1-Monooxygenase from Pseudomonas sp. Strain ATCC 29352

Abstract

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.