title: Unusual spectroscopic and ligand binding properties of the cytochrome P450-flavodoxin fusion enzyme XplA.
abstract: The Rhodococcus rhodochrous strain 11Y XplA enzyme is an unusual cytochrome P450-flavodoxin fusion enzyme that catalyzes reductive denitration of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX). We show by light scattering that XplA is a monomeric enzyme. XplA has high affinity for imidazole (K(d) = 1.6 μM), explaining previous reports of a red-shifted XplA Soret band in pure enzyme. The true Soret maximum of XplA is at 417 nm. Similarly, unusually weak XplA flavodoxin FMN binding (K(d) = 1.09 μM) necessitates its purification in the presence of the cofactor to produce hallmark flavin contributions absent in previously reported spectra. Structural and ligand-binding data reveal a constricted active site able to accommodate RDX and small inhibitory ligands (e.g. 4-phenylimidazole and morpholine) while discriminating against larger azole drugs. The crystal structure also identifies a high affinity imidazole binding site, consistent with its low K(d), and shows active site penetration by PEG, perhaps indicative of an evolutionary lipid-metabolizing function for XplA. EPR studies indicate heterogeneity in binding mode for RDX and other ligands. The substrate analog trinitrobenzene does not induce a substrate-like type I optical shift but creates a unique low spin EPR spectrum due to influence on structure around the distal water heme ligand. The substrate-free heme iron potential (-268 mV versus NHE) is positive for a low spin P450, and the elevated potential of the FMN semiquinone/hydroquinone couple (-172 mV) is also an adaptation that may reflect (along with the absence of a key Thr/Ser residue conserved in oxygen-activating P450s) the evolution of XplA as a specialized RDX reductase catalyst.
authors.affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Soi H
authors.initials: SH
authors.lastname: Bui
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: McLean
authors.forename: Myles R
authors.initials: MR
authors.lastname: Cheesman
authors.forename: Justin M
authors.initials: JM
authors.lastname: Bradley
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.forename: Colin W
authors.initials: CW
authors.lastname: Levy
authors.forename: David
authors.initials: D
authors.lastname: Leys
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D053834:Explosive Agents / Q000737:chemistry* / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry* / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D012240:Rhodococcus / Q000201:enzymology*; D014227:Triazines / Q000737:chemistry* / Q000378:metabolism
chemical_list: D053834:Explosive Agents; D005418:Flavodoxin; D007093:Imidazoles; D008024:Ligands; D014227:Triazines; C029899:imidazole; D003577:Cytochrome P-450 Enzyme System; C009160:cyclonite