A requirement for an active proton delivery network supports a compound I-mediated C–C bond cleavage in CYP51 catalysis

Tatiana Y Hargrove,Zdzislaw Wawrzak,F Peter Guengerich,Galina I Lepesheva

doi:10.1074/jbc.ra120.014064

Tatiana Y Hargrove, Zdzislaw Wawrzak + Show 2 more

Open Access

https://doi.org/10.1074/jbc.ra120.014064

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Abstract

CYP51 enzymes (sterol 14α-demethylases) are cytochromes P450 that catalyze multistep reactions. The CYP51 reaction occurs in all biological kingdoms and is essential in sterol biosynthesis. It removes the 14α-methyl group from cyclized sterol precursors by first forming an alcohol, then an aldehyde, and finally eliminating formic acid with the introduction of a Δ14-15 double bond in the sterol core. The first two steps are typical hydroxylations, mediated by an electrophilic compound I mechanism. The third step, C-C bond cleavage, has been proposed to involve either compound I (i.e. FeO3+) or, alternatively, a proton transfer-independent nucleophilic ferric peroxo anion (compound 0, i.e. Fe3+O2-). Here, using comparative crystallographic and biochemical analyses of WT human CYP51 (CYP51A1) and its D231A/H314A mutant, whose proton delivery network is destroyed (as evidenced in a 1.98-Å X-ray structure in complex with lanosterol), we demonstrate that deformylation of the 14α-carboxaldehyde intermediate requires an active proton relay network to drive the catalysis. These results indicate a unified, compound I-based mechanism for all three steps of the CYP51 reaction, as previously established for CYP11A1 and CYP19A1. We anticipate that our approach can be applied to mechanistic studies of other P450s that catalyze multistep reactions, such as C-C bond cleavage.

Highlights

Cytochrome P450 (CYP) enzymes are heme–thiolate coordinated monooxygenases that catalyze a vast variety of reactions involving xenobiotic and endogenous compounds
The asymmetric unit consisted of two P450 molecules, each of them revealing a clear electron density for one molecule of lanosterol bound within the sterol 14a-demethylase (CYP51) active site (Fig. 4A and Fig. S2)
The C3-OH group of the sterol nucleus is directed toward the substrate access channel entrance approaching b-strand 1–4 and forming an H-bond with the main-chain oxygen of Ile-379, the aliphatic arm occupies the deepest portion of the active site reaching helices C and I, and the C14a-methyl group is located 4 Å above the heme iron, indicative of a catalytically competent orientation

Summary

Edited by Ruma Banerjee

CYP51 enzymes (sterol 14a-demethylases) are cytochromes P450 that catalyze multistep reactions. A distinguishing difference between the two types of mechanisms is that the formation of compound I requires a specific proton relay network (to minimize the unwanted production of hydrogen peroxide via nonspecific proton donation to dioxygen in P450 enzymes) [23,24,25,26], whereas the ferric peroxo-anion abstracts hydrogens from substrates and does not depend on proton delivery [13] It has been generally accepted (going back to the 1980s [27–29]) that the proton delivery network involves a conserved P450 threonine in the I helix, a preceding charged residue, and a solvent-accessible residue(s) of opposite charge in the middle of helix F [3, 30, 31]. The D231A/H314A mutant is the first example of a CYP51 that, upon the addition of substrate, is found completely in the highspin form (Fig. 3), without structural evidence it has remained unclear whether it forms a stoichiometric

Crystallographic analysis

Data collection and refinement statistics

PDB code

Biochemical analysis

Conclusions

Protein expression and purification