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, 4,6,8,10,4,6,8,10, 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., mononitroso 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 doubledenitrated 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.

In vitro degradation of hexanitrohexaazaisowurtzitane (CL‐20) by cytosolic enzymes of Japanese quail and the rabbit

“…Gel analysis was carried out using the BioRad Quantity One Analysis software (BioRad). The N-terminal microsequencing was performed as described previously [11].…”

Catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate with a proteomics approach

“…The most straightforward approach to characterize degradation pathways is to map the catabolic enzymes directly involved. To this end, enzymes have been usually assayed, purified and characterized individually (Bhushan et al, 2005;Iwaki and Hasegawa, 2007;Krishnan et al, 2004;Lee et al, 2003). In recent years, with the completion of more and more genome sequencing projects, more and more research work on microbial proteomics projects has been carried out; accordingly, the number of publications on microbial proteomes has dramatically increased in the past few years.…”

Biodegradation kinetics of the nitramine explosive CL-20 in soil and microbial cultures

“…Most enzymes used for degradation are dehydrogenases which start degradation by transferring " e/H from NADH to CL-20 (Bhushan et al 2004(Bhushan et al , 2004a(Bhushan et al , 2005. It is well known that this reduction reaction is inhibited by O 2 because oxygen is a competing electron acceptor.…”

“…(Bhushan et al 2004a), nitroreductase from Escherichia coli (Bhushan et al 2004), dehydrogenase from Clostridium sp. (Bhushan et al 2005), and manganese peroxidase from white-rot fungus Nemalotoma frowardii (Fournier et al 2006).…”