title: Biotransformation of CL-20 by a dehydrogenase enzyme from Clostridium sp. EDB2.
abstract: 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 C(6)H(6)N(12)O(11), C(6)H(7)N(11)O(10), and C(6)H(6)N(10)O(8), respectively. Identity of all the three metabolites were confirmed by using ring-labeled [(15)N]CL-20 and the nitro-group-labeled [(15)NO(2)]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.
authors.affiliation: Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, 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: Jalal
authors.initials: J
authors.lastname: Hawari
mesh_terms: D000693:Anaerobiosis; D001372:Aza Compounds / Q000378:metabolism*; D001673:Biodegradation, Environmental; D001709:Biotechnology / Q000379:methods; D003013:Clostridium / Q000201:enzymology* / Q000254:growth & development; D006571:Heterocyclic Compounds / Q000378:metabolism*; D009573:Nitrites; D009609:Nitrous Oxide / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / 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; D009573:Nitrites; D010088:Oxidoreductases; D009609:Nitrous Oxide