4EP6

Crystal structure of the XplA heme domain in complex with imidazole and PEG

Summary for 4EP6

• Entry DOI: 10.2210/pdb4ep6/pdb
• Related: 2WIV 2WIY
• Descriptor: Cytochrome P450-like protein XplA, PROTOPORPHYRIN IX CONTAINING FE, IMIDAZOLE, ... (5 entities in total)
• Functional Keywords: p450 enzyme, p450 monoxygenase, explosive rdx, oxidoreductase
• Biological source: Rhodococcus rhodochrous
• Total number of polymer chains: 1
• Total formula weight: 44317.72

Authors

Bui, S.H.,McLean, K.J.,Cheesman, M.R.,Bradley, J.M.,Rigby, S.E.J.,Leys, D.,Munro, A.W. (deposition date: 2012-04-17, release date: 2012-05-09, Last modification date: 2023-09-13)

Primary citation

Bui, S.H.,McLean, K.J.,Cheesman, M.R.,Bradley, J.M.,Rigby, S.E.,Levy, C.W.,Leys, D.,Munro, A.W.
Unusual Spectroscopic and Ligand Binding Properties of the Cytochrome P450-Flavodoxin Fusion Enzyme XplA.
J.Biol.Chem., 287:19699-19714, 2012

PubMed 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.

PubMed: 22500029
DOI: 10.1074/jbc.M111.319202

Experimental method

X-RAY DIFFRACTION (2.3 Å)