Hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX) is a military explosive that has become a recalcitrant environmental pollutant over the last few decades owing to its production, storage and use. CYP177A1 (XplA) is a biotechnologically interesting and novel class of P450-flavodoxin fusion enzyme identified from Rhodococcus rhodochrous strain 11Y that catalyses the breakdown of RDX. Its redox partner is a NAD(P)H-dependent FAD-binding flavodoxin reductase (XplB). This study reports the biochemical, biophysical and structural properties of these two enzymes which form a novel P450 redox system with unique domain organisation. These reveal novel features for a P450 enzyme with non-standard UV/Visible spectroscopic features and unusual ligand binding properties. Unexpectedly, XplA's affinity for imidazole is exceptionally high (Kd = 1.57 μM), explaining previous reports of a red- shifted XplA Soret band in pure enzyme. XplA's true Soret maximum is at 417 nm. Similarly, the XplA flavodoxin domain displays unusually weak FMN binding (Kd = 1.09 μM), necessitating its reconstitution with the FMN cofactor. Ligand binding data demonstrate XplA's constricted active site, which can only accommodate RDX and small inhibitory ligands (e.g. 4-phenylimidazole and morpholine) while discriminating against larger azole drugs. The crystal structure identifies a high affinity imidazole binding site, consistent with its low Kd, and shows active site penetration by PEG, perhaps indicative of an evolutionary lipid metabolising function for XplA. The substrate-free heme iron potential (-268 mV vs. NHE) is positive for a low spin P450, consistent with the predominantly reductive role of XplA. The elevated potential of the FMN semiquinone/hydroquinone couple (-172 mV) is also consistent with this functional adaptation. The XplB reductase partner could not be isolated with the FAD cofactor incorporated to make holoprotein. However, the protein was isolated in a soluble and homogenous state which demonstrated very weak FAD affinity. XplB's ability to interact with XplA and a pyridine nucleotide coenzyme was demonstrated, indicating the enzyme was functional in the presence of FAD. XplA's unusual molecular selectivity, structural and thermodynamic properties likely reflect its evolution as a specialised RDX reductase catalyst.
|Date of Award||1 Aug 2012|
- The University of Manchester
|Supervisor||Andrew Munro (Supervisor)|