Mechanism of O2-dependent Nitramine Degradation by a Heme Enzyme
Abstract
The nitramine functional group (RÐN(H)NO2) is responsible for the explosive nature of the compounds RDX, CL-20, and HMX that are found in military-grade munitions and ordnance. These compounds are human toxins that contaminate soil and drinking water as a result of detonation or munitions production. Some microorganisms degrade these nitramine compounds and use the breakdown products as nitrogen and carbon sources. These microbially-mediated decomposition pathways could be useful to remediate contamination sites. Characterization of the molecular mechanisms of these degradations are critical for assessing the safety of the decomposition products and for improving the efficiency of remediation strategies. Two bacterial heme-containing enzymes have been identified that catalyze nitramine decompositions. However, neither of their molecular mechanisms are characterized. The cyclic nitramine RDX has been shown to be degraded by a cytochrome P450 homolog called XplA. In the presence of dioxygen (O2), nitramine decomposition by XplA results in formation of formaldehyde, nitrite, and a linear nitramine 4-nitro-2,4,-diazabutanal (NDAB). The complex product distribution complicates elucidating the mechanisms solely from these product data. Additionally, the product distribution changes in the absence of O2; methylenenitramine is the major product instead of NDAB and less nitrite is produced. While these are important observations, more work needs to be done to understand the nitramine decomposition pathway. A second enzyme called NnlA provides a simpler route to isolate the decomposition chemistry of the nitramine functional group. NnlA catalyzes the decomposition of a simple nitramine called N-nitroglycine (NNG) to form glyoxylate, nitrite, and a second, as yet, unidentified nitrogenous product. The observation of glyoxylate as a product strongly suggests there is little rearrangement or fragmentation of the NNG backbone and the decomposition chemistry occurs mostly at the nitramine functionality. This combined with the less complex product distribution suggest NNG as an ideal model to study enzymatic nitramine decomposition mechanisms. There are several gaps in the knowledge of NnlA that need to be addressed: 1) the structure of the active and substrate binding sites, 2) the full reaction stoichiometry, 3) if nitramine decomposition by NnlA is O2 dependent, and 4) the role of the heme. Structural, biochemical, and mechanistic studies are proposed to address these knowledge gaps, with an emphasis on trapping and characterizing enzyme-bound nitramine decomposition intermediates. Furthermore, a strategy to broaden the NnlA substrate scope is described using rationale engineering and directed evolution approaches. Completion of the work will inform remediation strategies and expand the substrate scope of NnlA to complement existing remediation strategies for RDX, HMX, and CL-20 and enable selective sensing of these compounds. The emphasis on trapping intermediates of nitramine degradation will ensure broader relevance of the work to other nitramine decomposition pathways, such as detonation, where intermediate trapping is more difficult.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Aug 31, 2020
- Source ID
- W911NF2010286
Entities
People
- Jonathan D Caranto
Organizations
- Army Contracting Command
- Office of the Secretary of Defense
- University of Central Florida