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Nitroreductase catalyzed biotransformation of CL-20
Bharat Bhushan1, Annamaria Halasz, Jalal Hawari
1Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2.
Biochemical and Biophysical Research Communications
|August 18, 2004
Summary
Nitroreductase from Escherichia coli biotransforms CL-20, producing a metabolite that decomposes into glyoxal and formic acid. This process involves N-denitration and is faster under anaerobic conditions, with FMN-site involvement suggested.
Area of Science:
- Biochemistry
- Environmental Microbiology
- Chemical Engineering
Background:
- Previous studies showed Pseudomonas sp. ATCC 29352 biotransformed CL-20 into a metabolite that decomposed into N2O, NH4+, and HCOOH.
- CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane) is a high-energy material with potential environmental implications.
Purpose of the Study:
- To investigate the biotransformation of CL-20 by nitroreductase from Escherichia coli.
- To identify the intermediate metabolite and its decomposition products.
- To determine the kinetics and stoichiometry of the biotransformation process.
Main Methods:
- Enzymatic assays using nitroreductase from Escherichia coli with CL-20 as substrate.
- Analysis of reaction products using mass spectrometry and chemical identification.
- Kinetic studies under anaerobic and aerobic conditions.
- Stoichiometric analysis of reaction products and mass balance calculations.
Main Results:
- Nitroreductase catalyzed a one-electron transfer to CL-20, forming a radical anion and an N-denitrated metabolite (C6H6N10O8).
- The metabolite was identified as 1,4,5,8-tetranitro-1,3a,4,4a,5,7a,8,8a-octahydro-diimidazo[4,5-b:4',5'-e]pyrazine, which decomposed to glyoxal and formic acid.
- Biotransformation rates were significantly higher under anaerobic (3.4 nmol/min/mg) than aerobic (0.25 nmol/min/mg) conditions.
- Product stoichiometry indicated the formation of nitrite, nitrous oxide, formic acid, glyoxal, and ammonium ions.
Conclusions:
- Escherichia coli nitroreductase effectively biotransforms CL-20 via N-denitration, yielding glyoxal and formic acid.
- The flavin mononucleotide (FMN)-binding site of nitroreductase is likely involved in CL-20 biotransformation.
- Understanding these pathways is crucial for developing bioremediation strategies for CL-20 contaminated environments.