Exploring the biochemical properties and remediation applications of the unusual explosive-degrading P450 system XplA/B

Abstract

Widespread contamination of land and groundwater has resulted from the use, manufacture, and storage of the military explosive hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX). This contamination has led to a requirement for a sustainable, low-cost method to remediate this problem. Here, we present the characterization of an unusual microbial P450 system able to degrade RDX, consisting of flavodoxin reductase XplB and fused flavodoxin-cytochrome P450 XplA. The affinity of XplA for the xenobiotic compound RDX is high (K d ‫؍‬ 58 M) and comparable with the Km of other P450s toward their natural substrates (ranging from 1 to 500 M). The maximum turnover (kcat) is 4.44 per s, only 10-fold less than the fastest self-sufficient P450 reported, BM3. Interestingly, the presence of oxygen determines the final products of RDX degradation, demonstrating that the degradation chemistry is flexible, but both pathways result in ring cleavage and release of nitrite. Carbon monoxide inhibition is weak and yet the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a potent inhibitor. To test the efficacy of this system for the remediation of groundwater, transgenic Arabidopsis plants expressing both xplA and xplB were generated. They are able to remove saturating levels of RDX from liquid culture and soil leachate at rates significantly faster than those of untransformed plants and xplA-only transgenic lines, demonstrating the applicability of this system for the phytoremediation of RDX-contaminated sites.cytochrome P450 ͉ phytoremediation ͉ hexa-hydro-1,3,5-trinitro-1,3,5-triazine

The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

“…The products isolated from XplA-XplB-catalyzed degradation of RDX in vitro were shown to be different under aerobic and anaerobic conditions after initial denitration to imine intermediate 2 ( Fig. 1) (5). Under anaerobic conditions A, 2 would undergo hydration to give intermediate 4.…”

“…The products of biotransformation of RDX by this bacterium were shown to be nitrite and formaldehyde. A gene cluster in strain 11Y essential for RDX degradation was identified and shown to contain a novel cytochrome P450, termed XplA, and a redox partner, XplB, which were shown together to be capable of catalyzing the biotransformation of RDX in vitro (5). Near identical xplA and xplB genes have now been identified in strains within the Actinomycetales isolated from geographically distinct sites (6).…”

“…Interestingly, these genes have been found to be plasmid encoded providing compelling evidence for recent lateral gene transfer. In the interests of developing an efficient technology for the targeted phytoremediation of RDX, the XplA-XplB system was expressed in transgenic strains of Arabidopsis thaliana and shown to successfully remediate RDX from contaminated soil (5,7).…”

Arabidopsis Glutathione Transferases U24 and U25 Exhibit a Range of Detoxification Activities with the Environmental Pollutant and Explosive, 2,4,6-Trinitrotoluene

“…One way this problem might be tackled is through the use of plants that are adapted to detoxify these compounds. This could be achieved either by traditional breeding programs or by genetic modification, as has been demonstrated previously for both RDX and TNT (Hannink et al, 2001;Rylott et al, 2006;Jackson et al, 2007).In the majority of species tested so far ( (Schoenmuth and Pestemer, 2004), TNT is located almost entirely in the roots (Sens et al, 1998(Sens et al, , 1999Hannink et al, 2007;Van Dillewijn et al, 2008;Brentner et al, 2010). Endogenous metabolism of TNT by plants has been characterized Rylott et al, 2011b), with recent research focusing on the model plant species…”

“…One way this problem might be tackled is through the use of plants that are adapted to detoxify these compounds. This could be achieved either by traditional breeding programs or by genetic modification, as has been demonstrated previously for both RDX and TNT (Hannink et al, 2001;Rylott et al, 2006;Jackson et al, 2007).…”