4ep6
Crystal structure of the XplA heme domain in complex with imidazole and PEGCrystal structure of the XplA heme domain in complex with imidazole and PEG
Structural highlights
Publication Abstract from PubMedThe 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 (Kd = 1.6 microM), explaining previous reports of a red-shifted XplA Soret band in pure enzyme. XplA's true Soret maximum is at 417 nm. Similarly, unusually weak XplA flavodoxin FMN binding (Kd = 1.09 microM) necessitates its purification in 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 Kd, 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 analogue 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 vs. 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 absence of a key Thr/Ser residue conserved in oxygen activating P450s) XplA's evolution as a specialized RDX reductase catalyst. Unusual spectroscopic and ligand binding properties of the cytochrome P450-flavodoxin fusion enzyme XplA.,Bui SH, McLean KJ, Cheesman MR, Bradley JM, Rigby SE, Levy CW, Leys D, Munro AW J Biol Chem. 2012 Apr 12. PMID:22500029[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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