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

Abstract

XplA is a cytochrome P450 of unique structural organization, consisting of a heme-domain that is C-terminally fused to its native flavodoxin redox partner. XplA, along with flavodoxin reductase XplB, has been shown to catalyze the breakdown of the nitramine explosive and pollutant hexahydro-1,3,5-trinitro-1,3,5-triazine (royal demolition explosive) by reductive denitration. The structure of the heme domain of XplA (XplA-heme) has been solved in two crystal forms: as a dimer in space group P2 1 to a resolution of 1.9 Å and as a monomer in space group P2 1 2 1 2 to a resolution of 1.5 Å , with the ligand imidazole bound at the heme iron. Although it shares the overall fold of cytochromes P450 of known structure, XplA-heme is unusual in that the kinked I-helix that traverses the distal face of the heme is broken by Met-394 and Ala-395 in place of the well conserved Asp/Glu plus Thr/Ser, important in oxidative P450s for the scission of the dioxygen bond prior to substrate oxygenation. The heme environment of XplA-heme is hydrophobic, featuring a cluster of three methionines above the heme, including Met-394. Imidazole was observed bound to the heme iron and is in close proximity to the side chain of Gln-438, which is situated over the distal face of the heme. Imidazole is also hydrogenbonded to a water molecule that sits in place of the threonine side-chain hydroxyl exemplified by Thr-252 in Cyt-P450cam. Both Gln-438 3 Ala and Ala-395 3 Thr mutants of XplA-heme displayed markedly reduced activity compared with the wild type for royal demolition explosive degradation when combined with surrogate electron donors. Royal demolition explosive (RDX)2 or cyclotrimethylenetrinitramine 1 (see Fig. 1) is a widely used explosive compound with both military and civil applications. The extensive global usage of RDX has resulted in concerns over environmental contamination, because it is both recalcitrant to degradation, leading to contamination in soil and ground water, and a potent convulsant and possible carcinogen. The bioremediation of RDX has thus been the focus of increasing research in recent years, with a number of bacterial strains reported to catalyze its degradation (1-3). Among these, Bruce and co-workers, using a selective enrichment technique, isolated Rhodococcus rhodochrous strain 11Y from an RDX-contaminated site, which was able to grow on RDX as the sole nitrogen source (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 intere...

Unusual Spectroscopic and Ligand Binding Properties of the Cytochrome P450-Flavodoxin Fusion Enzyme XplA

“…Thus, the XplA monomer probably interacts with its NAD(P)H-dependent XplB partner flavoprotein reductase in a 1:1 complex. The crystal structure for the XplA heme domain reported here is consistent with the earlier reported structure in terms of overall fold and active site organization (24). Close inspection of the distal pocket enables rationalization of the reason for the extraordinarily high affinity for imidazole.…”

“…In previous studies of XplA (formally CYP177A1), Bruce and co-workers (24) have expressed and purified the enzyme and have solved the crystal structure for its P450 (heme) domain (residue 154 to the end of the 552-amino acid flavocytochrome). Reductive denitration of RDX nitro groups was observed in turnover studies performed using the XplA/XplB combination.…”

Analysis of the xplAB -Containing Gene Cluster Involved in the Bacterial Degradation of the Explosive Hexahydro-1,3,5-Trinitro-1,3,5-Triazine

“…Together, the encoded P450 system catalyzes the reductive denitration of RDX, resulting in subsequent ring cleavage under aerobic and anaerobic conditions (9,10). Structural analyses, including X-ray studies (11), have revealed features not previously seen in cytochromes P450, including the fusion of the heme domain to a flavodoxin redox partner (12).…”

Role of Nitrogen Limitation in Transformation of RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine) by Gordonia sp. Strain KTR9

“…Microbially mediated RDX biodegradation has been reported under a number of conditions (5)(6)(7)(8)(9)(10)(11)(12)(13)(14), with only a few genes implicated in RDX degradation (15)(16)(17). Of these, the cytochrome P450 system encoded by xplAB is the best characterized (18,19).…”