title: Biotransformation of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) by denitrifying Pseudomonas sp. strain FA1.
abstract: The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 +/- 0.1 nmol h(-1) mg of cell biomass(-1) and 11.5 +/- 0.4 nmol h(-1) mg of protein(-1), respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO(2)(-)), 1.5 molecules of nitrous oxide (N(2)O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.
authors.affiliation: Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada.
authors.forename: Bharat
authors.initials: B
authors.lastname: Bhushan
authors.forename: Louise
authors.initials: L
authors.lastname: Paquet
authors.forename: Jim C
authors.initials: JC
authors.lastname: Spain
authors.forename: Jalal
authors.initials: J
authors.lastname: Hawari
mesh_terms: D001711:Biotransformation; D001952:Bridged-Ring Compounds / Q000737:chemistry* / Q000493:pharmacokinetics*; D002462:Cell Membrane / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D009243:NAD / Q000378:metabolism; D009574:Nitro Compounds / Q000737:chemistry* / Q000493:pharmacokinetics*; D011549:Pseudomonas / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001952:Bridged-Ring Compounds; D009574:Nitro Compounds; D009243:NAD; C083639:hexanitrohexaazaisowurzitane