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

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 XpIB and fused flavodoxin-cytochrome P450 XpIA. The affinity of XpIA for the xenobiotic compound RDX is high (K-d = 58 mu M) and comparable with the K. of other P450s toward their natural substrates (ranging from 1 to 500 mu M). The maximum turnover (k(cat)) 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 xpIA and xpIB 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 xpIA-only transgenic lines, demonstrating the applicability of this system for the phytoremediation of RDX-contaminated sites.