Biotransformation of CL-20 by a dehydrogenase enzyme from Clostridium sp. EDB2

In a previous study, a marine isolate Clostridium sp. EDB2 degraded 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) under anaerobic conditions (Bhushan B, Halasz A, Thiboutot S, Ampleman G, Hawari J (2004c) Chemotaxis-mediated biodegradation of cyclic nitramine explosives RDX, HMX, and CL-20 by Clostridium sp. EDB2. Biochem Biophys Res Commun 316:816–821); however, the enzyme responsible for CL-20 degradation was not known. In the present study, we isolated and purified an enzyme, from strain EDB2, responsible for CL-20 degradation. The enzyme was membrane-associated and NADH-dependent and had a molecular weight of 56 kDa (with SDS-PAGE). N-terminal amino acid sequence of enzyme revealed that it belonged to dehydrogenase class of enzymes. The purified enzyme degraded CL-20 at a rate of 18.5 nmol/h mg protein under anaerobic conditions. Carbon and nitrogen mass balance of the products were 100 and 64%, respectively. In LC–MS–MS studies, we detected three different initial metabolites from CL-20, i.e., mono-nitroso derivative, denitrohydrogenated product, and double-denitrated isomers with molecular weight of 422, 393, and 346 Da, corresponding to presumed empirical formulas of C₆H₆N₁₂O₁₁, C₆H₇N₁₁O₁₀, and C₆H₆N₁₀O₈, respectively. Identity of all the three metabolites were confirmed by using ring-labeled [¹⁵N]CL-20 and the nitro-group-labeled [¹⁵NO₂]CL-20. Taken together, the above data suggested that the enzyme degraded CL-20 via three different routes: Route A, via two single electron transfers necessary to release two nitro-groups from CL-20 to produce two double-denitrated isomers; Route B, via a hydride transfer necessary to produce a denitrohydrogenated product; and Route C, via transfer of two redox equivalents to CL-20 necessary to produce a mono-nitroso derivative of CL-20. This is the first biochemical study which showed that CL-20 degradation can be initiated via more than one pathway.