Homodimerization Counteracts the Detrimental Effect of Nitrogenous Heme Ligands on the Enzymatic Activity of Acanthamoeba castellanii CYP51

Abstract

Acanthamoeba castellanii is a free-living amoeba that can cause severe eye and brain infections in humans. At present, there is no uniformly effective treatment for any of these infections. However, sterol 14α-demethylases (CYP51s), heme-containing cytochrome P450 enzymes, are known to be validated drug targets in pathogenic fungi and protozoa. The catalytically active P450 form of CYP51 from A. castellanii (AcCYP51) is stabilized against conversion to the inactive P420 form by dimerization. In contrast, Naegleria fowleri CYP51 (NfCYP51) is monomeric in its active P450 and inactive P420 forms. For these two CYP51 enzymes, we have investigated the interplay between the enzyme activity and oligomerization state using steady-state and time-resolved UV–visible absorption spectroscopy. In both enzymes, the P450 → P420 transition is favored under reducing conditions. The transition is accelerated at higher pH, which excludes a protonated thiol as the proximal ligand in P420. Displacement of the proximal thiolate ligand is also promoted by adding exogenous nitrogenous ligands (N-ligands) such as imidazole, isavuconazole, and clotrimazole that bind at the opposite, distal heme side. In AcCYP51, the P450 → P420 transition is faster in the monomer than in the dimer, indicating that the dimeric assembly is critical for stabilizing thiolate coordination to the heme and thus for sustaining AcCYP51 activity. The spectroscopic experiments were complemented with size-exclusion chromatography and X-ray crystallography studies. Collectively, our results indicate that effective inactivation of the AcCYP51 function by azole drugs is due to synergistic interference with AcCYP51 dimerization and promoting irreversible displacement of the proximal heme–thiolate ligand.

Miconazole-like Scaffold is a Promising Lead for Naegleria fowleri-Specific CYP51 Inhibitors

“…A plausible explanation of this phenomenon is irreversible deterioration of the heme Fe thiolate bond upon binding of azole inhibitors, as we demonstrated elsewhere by UV–vis spectroscopy for CYP51 of N. fowleri and Acanthamoeba castellanii . 53 Depending on experimental conditions, deterioration rates are on a minute-to-hours time scale that is consistent with protein conformational motions. The broad Soret band indicates an enormous structural heterogeneity and flexibility of the heme pocket; close inspection of the spectra suggests that multiple species are present.…”

“…Depending on experimental conditions, deterioration rates are on a minute-to-hours time scale that is consistent with protein conformational motions. The broad Soret band indicates an enormous structural heterogeneity and flexibility of the heme pocket; close inspection of the spectra suggests that multiple species are present …”

“…The broad Soret band indicates an enormous structural heterogeneity and flexibility of the heme pocket; close inspection of the spectra suggests that multiple species are present. 53 …”

“…A plausible explanation of this phenomenon is irreversible deterioration of the heme Fe thiolate bond upon binding of azole inhibitors, as we demonstrated elsewhere by UV−vis spectroscopy for CYP51 of N. fowleri and Acanthamoeba castellanii. 53 Depending on among existing azole drugs, miconazole molecular scaffolds were singled out as the most complementary to the NfCYP51 active site. After 1 h of drug exposure, blood and brain samples were collected and analyzed by means of HPLC-ESI-MS/MS.…”