Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

Oxygen‐18 Labeling Reveals a Mixed Fe−O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α‐Demethylation

“…The synthesis of deuterated 14α-formyl 24,25-dihydrolanosterol (14α-CDO dihydrolanosterol) was based on work of Morisaki and associates, [26] which began with commercial 7-dehydrocholesterol and generated the protiated product in 14 steps to give the desired 14α-aldehyde. [27] The partially deuterated aldehyde was generated in three more steps by sequential Dess-Martin periodinane oxidation, [28] reduction with NaBD 4 , Dess-Martin periodinane oxidation, and Scheme 2. Oxidation of 24,25-dihydrolanosterol to 24,25-dihydro-4.4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol (24,25-dihydro FF-MAS) and formic acid.…”

“…[27] As our 18 O labeling data clearly indicated the dominance of P450 Compound 0 (Figure 4), we were interested as to whether the proposed 14α-formyl lanosterol derivative (Baeyer-Villiger intermediate) was also a product under our experimental conditions, namely, utilizing a purified recombinant enzyme system (devoid of esterases in microsomes, which could degrade such an ester intermediate) and on-line LC-MS (which was not available to Fischer et al [12c] ). We performed a 14α-CDO dihydrolanosterol deformylation assay under steady-state conditions [27] and subjected the products of the reaction to reversed-phase (C 18 ) HPLC coupled to electrospray ionization (ESI) MS analysis. Our analysis revealed an NADPH-dependent chromatographic peak (t R 3.05 min) eluting as an early shoulder on the substrate peak (t R 3.51 min), corresponding to the chromatographic migration of the putative Baeyer-Villiger intermediate in the earlier report (Figures 6, S8).…”

“…[24] We had previously reported that the overall three-step reaction was highly kinetically processive, in that lanosterol and reaction intermediates are bound tightly to the enzyme and driven to final product formation. [27] Accordingly, we investigated whether the binding mode of the aldehyde (for CÀ C cleavage) was similar to the binding mode of lanosterol (for hydroxylation) and crystallized the enzyme in complex with our synthetic aldehyde intermediate for comparison.…”

“…This is in line with the finding that the three-step reaction of sterol 14α-demethylation is a highly kinetically processive process. [27]…”

Oxygen-18 Labeling Defines a Ferric Peroxide (Compound 0) Mechanism in the Oxidative Deformylation of Aldehydes by Cytochrome P450 2B4

“…The 18 O labeling approach that we have employed here is applicable to other P450-catalyzed oxidative deformylations, e.g., P450 19A1-catalyzed androstenedione aromatization (vide supra) and P450 51-catalyzed lanosterol 14α-demethylation. , Although Ortiz de Montellano and co-workers employed 18 O 2 to study an oxidative terminal side chain carbon cleavage reaction of cholesterol by bacterial P450 125A1, direct evidence of Compound 0 contribution was not presented, and this question could also be addressed by employing deuterium-labeled formic acid analysis. In principle, the approach can be applied to C–C bond scissions that yield acetic acid (e.g., P450 17A1-catalyzed 17α,20-lyase reaction and P450 1A2-catalyzed side-chain cleavage of nabumetone), , but with α-ketols, the 18 O labeling results are not unambiguous. ,, …”