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

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. 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. Cytochrome P450 (CYP) enzymes are heme–thiolate coordinated monooxygenases that catalyze a vast variety of reactions involving xenobiotic and endogenous compounds. CYP51 (sterol 14α-demethylases) play a crucial role in the production of sterols in all phyla and are important drug targets (1Lepesheva G.I. Friggeri L. Waterman M.R. CYP51 as drug targets for fungi and protozoan parasites: past, present and future.Parasitology. 2018; 145 (29642960): 1820-183610.1017/S0031182018000562Crossref PubMed Scopus (41) Google Scholar, 2Friggeri L. Hargrove T.Y. Wawrzak Z. Guengerich F.P. Lepesheva G.I. Validation of human sterol 14α-demethylase (CYP51) druggability: structure-guided design, synthesis, and evaluation of stoichiometric, functionally irreversible inhibitors.J. Med. Chem. 2019; 62 (31663733): 10391-1104010.1021/acs.jmedchem.9b01485Crossref PubMed Scopus (5) Google Scholar, 3Lepesheva G.I. Waterman M.R. Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms.Biochim. Biophys. Acta. 2007; 1770 (16963187): 467-47710.1016/j.bbagen.2006.07.018Crossref PubMed Scopus (273) Google Scholar). The CYP51 reaction includes three steps (Fig. 1) (i.e. three cytochrome P450 catalytic cycles [Fig. 2]); the first two steps are formal hydroxylations, and the third step is a C–C bond cleavage.Figure 2The cytochrome P450 catalytic cycle. Binding of the substrate (1) displaces the water molecule from the sixth (distal) coordination site of the ferric heme iron, changing its spin state from hexa-coordinated low to penta-coordinated high, increasing its redox potential, and facilitating the first electron transfer from the protein redox partner (2). The ferrous iron complex binds molecular oxygen (3), which triggers acceptance of the second electron producing the nucleophilic ferric peroxo anion (4) (compound 0, or Fe3+–O2−). The ferric peroxo complex is then protonated to form the ferric hydroperoxo state (5), the second protonation of the distal oxygen atom causing the O-O bond scission, release of a water molecule, and generation of a highly reactive electrophilic Fe4+-oxo cation radical (FeO3+, or compound I) (6). The compound I mechanism requires two protons delivered from the protein surface to the iron-bound dioxygen via the active proton delivery network. In the compound 0 mechanism, the dioxygen abstracts protons from the substrate, forming a peroxo-hemiacetal intermediate and, thus, being independent of proton delivery.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Whereas it is now generally agreed that most P450 reactions use compound I (4Rittle J. Green M.T. Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics.Science. 2010; 330 (21071661): 933-93710.1126/science.1193478Crossref PubMed Scopus (851) Google Scholar) (step 6 in Fig. 2, FeO3+) as the active iron species, the C–C bond cleavage (lyase) mechanism in the multistep reactions is still under debate (5Guengerich F.P. Yoshimoto F.K. Formation and cleavage of C-C bonds by enzymatic oxidation-reduction reactions.Chem. Rev. 2018; 118 (29932643): 6573-666510.1021/acs.chemrev.8b00031Crossref PubMed Scopus (80) Google Scholar). 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A residue is a in CYP51 an residue in other and a in the is in CYP51 in other In the and CYP51 structures all a and the of an are in and in human CYP51 as we binding of the substrate to the T. CYP51 a conformational that the the to in proton delivery T.Y. Wawrzak Z. S.A. Guengerich F.P. Waterman M.R. Lepesheva G.I. Binding of a physiological substrate conformational in cytochrome P450 Biol. Chem. 2018; 293 Full Text Full Text PDF PubMed Scopus Google Scholar). of of the in human CYP51 catalysis of with the double a in of more two of in the and complex formation upon or substrate binding of the but the of the I to lanosterol spin state in the heme in the WT D231A/H314A T.Y. Wawrzak Z. S.A. Guengerich F.P. Waterman M.R. Lepesheva G.I. Binding of a physiological substrate conformational in cytochrome P450 Biol. Chem. 2018; 293 Full Text Full Text PDF PubMed Scopus Google Scholar). The D231A/H314A is the first of a CYP51 upon the of substrate, is in the form (Fig. although structural evidence it has it a complex in or the heme iron the to in the form the In we the 1.98-Å X-ray structure of the human CYP51 D231A/H314A in complex with lanosterol and applied comparative crystallographic and biochemical analyses of and WT human enzymes to the CYP51 mechanism. We that the activity of the the substrate is as as its activity with to the that proton transfer is for the third step of the CYP51 reaction and compound I is the active The complex was in the and the final structure was to 1.98-Å with an of and of The and structure have been in the under The of two P450 of a electron for molecule of lanosterol the CYP51 active site (Fig. and Fig. and human of of from in of of in a new The binding of lanosterol in the human structure was to be the as the binding of in the T. CYP51 T.Y. Wawrzak Z. S.A. Guengerich F.P. Waterman M.R. Lepesheva G.I. Binding of a physiological substrate conformational in cytochrome P450 Biol. Chem. 2018; 293 Full Text Full Text PDF PubMed Scopus Google Scholar) (Fig. and The group of the sterol is the substrate and forming an with the oxygen of to in T. the the of the active site and and the group is 4 the heme iron, of a The of both protozoan and human CYP51 enzymes are and and so is the conformational in the T. CYP51 the P450 in the in that three are and is and human CYP51 (Fig. The involve most of the structural of the CYP51 active The of the and the of the to a with and the side-chain of of the active site is to that of in T. the residue we have to in the electron transfer T.Y. Wawrzak Z. S.A. Guengerich F.P. Waterman M.R. Lepesheva G.I. Binding of a physiological substrate conformational in cytochrome P450 Biol. Chem. 2018; 293 Full Text Full Text PDF PubMed Scopus Google Scholar). In the with the heme is and a group is now the P450 surface (Fig. and Fig. The of the of I (Fig. and Fig. the the substrate and the between the of and In the human CYP51 mutant, two are now are in the of the transfer of protons from solvent to the iron-bound dioxygen for the formation of compound I WT human CYP51 and the D231A/H314A with of the presence of the substrate, and the P450 from the difference of the to the from the The of enzymatic with via was by the presence of the substrate, in the in the WT whereas lanosterol the of not in either (Fig. These results support the that a substrate-induced conformational does in and the enzymatic of the WT is because of a in the of by the WT (Fig. because WT CYP51 lanosterol the was In the of the substrate, the of for WT CYP51 and the mutant, both of the structural and biochemical data indicate that the D231A/H314A not the of human CYP51 to substrate, with the electron protein and The of that the proton is the for the of enzymatic The a to the mechanism of the C–C bond cleavage step in CYP51 catalysis is by a nucleophilic peroxo it not protons from the and the of the D231A/H314A to catalyze deformylation of the 14α-carboxaldehyde intermediate of lanosterol (as to its to be with or that of the reaction occurs via compound for which the formation transfer of protons from the solvent is (Fig. the of the D231A/H314A to catalyze deformylation of the 14α-carboxaldehyde intermediate We that catalytic turnover of the WT with the intermediate as the substrate of was as as that with lanosterol of which is in that the reaction occurs in final step of three of in The activity of the was as in the of lanosterol of (Fig. the reaction with being low to T.Y. Wawrzak Z. S.A. Guengerich F.P. Waterman M.R. Lepesheva G.I. Binding of a physiological substrate conformational in cytochrome P450 Biol. Chem. 2018; 293 Full Text Full Text PDF PubMed Scopus Google Scholar) and reaction for to be The data that all three steps of CYP51 catalysis have the compound mechanism. It has been proposed that for a C–C bond cleavage reaction to the ferric peroxo oxidant of compound a P450 substrate a group to the which was to be the for but not for CYP19A1 P.J. Duggal R. Denisov I.G. Gregory M.C. Sligar S.G. Kincaid J.R. Human cytochrome CYP17A1: the structural basis for compromised lyase activity with 17-hydroxyprogesterone.J. Am. Chem. Soc. 2018; 140 (29758981): 7324-733110.1021/jacs.8b03901Crossref PubMed Scopus (15) Google Scholar, P.J. Luthra A. Sligar S.G. Kincaid J.R. Resonance Raman spectroscopy of the oxygenated intermediates of human CYP19A1 implicates a compound I intermediate in the final lyase sep.J. Am. Chem. Soc. 2014; 136 (24645879): 4825-482810.1021/ja500054cCrossref PubMed Scopus (38) Google Scholar). is also not the for as in the CYP51 substrates the group the is from the group and with the oxygen in the (Fig. the evidence compound I mechanisms in three of the steroid C–C bond cleavage reactions (Fig. and Fig. It be that in CYP17A1, of the evidence suggests the of the substrate group with compound I (5Guengerich F.P. Yoshimoto F.K. Formation and cleavage of C-C bonds by enzymatic oxidation-reduction reactions.Chem. Rev. 2018; 118 (29932643): 6573-666510.1021/acs.chemrev.8b00031Crossref PubMed Scopus (80) Google Scholar, 16Gonzalez E. Johnson K.M. Pallan P.S. Phan T.T.N. Zhang W. Lei L. Wawrzak Z. Yoshimoto F.K. Egli M. Guengerich F.P. Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes.J. Biol. Chem. 2018; 293 (29212707): 541-55610.1074/jbc.RA117.000504Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). in the of CYP17A1, the 18O labeling is not in discerning between the two (5Guengerich F.P. Yoshimoto F.K. Formation and cleavage of C-C bonds by enzymatic oxidation-reduction reactions.Chem. Rev. 2018; 118 (29932643): 6573-666510.1021/acs.chemrev.8b00031Crossref PubMed Scopus (80) Google Scholar, F.K. Gonzalez E. Auchus R.J. Guengerich F.P. Mechanism of 17α,20-lyase and new hydroxylation reactions of human cytochrome P450 17A1: 18O labelling and oxygen surrogate evidance for a role of a perferryl oxygen.J. Biol. Chem. 2016; 291 (27339894): 17143-1716410.1074/jbc.M116.732966Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and the intermediates have not been to be (5Guengerich F.P. Yoshimoto F.K. Formation and cleavage of C-C bonds by enzymatic oxidation-reduction reactions.Chem. Rev. 2018; 118 (29932643): 6573-666510.1021/acs.chemrev.8b00031Crossref PubMed Scopus (80) Google Scholar, P.J. Gregory M.C. Denisov I.G. Sligar S.G. Kincaid J.R. Unveiling the crucial intermediates in androgen production.Proc. Natl. Acad. Sci. U S A. 2015; 112 (26668369): 15856-1586110.1073/pnas.1519376113Crossref PubMed Scopus (42) Google Scholar, 14Mak P.J. Duggal R. Denisov I.G. Gregory M.C. Sligar S.G. Kincaid J.R. Human cytochrome CYP17A1: the structural basis for compromised lyase activity with 17-hydroxyprogesterone.J. Am. Chem. Soc. 2018; 140 (29758981): 7324-733110.1021/jacs.8b03901Crossref PubMed Scopus (15) Google Scholar, P.J. Luthra A. Sligar S.G. Kincaid J.R. Resonance Raman spectroscopy of the oxygenated intermediates of human CYP19A1 implicates a compound I intermediate in the final lyase sep.J. Am. Chem. Soc. 2014; 136 (24645879): 4825-482810.1021/ja500054cCrossref PubMed Scopus (38) Google Scholar), so more experimental on is in the of P450 reactions that can be to compound 0 ferric peroxo anion C–C bond cleavage and other types of has (5Guengerich F.P. Yoshimoto F.K. Formation and cleavage of C-C bonds by enzymatic oxidation-reduction reactions.Chem. Rev. 2018; 118 (29932643): 6573-666510.1021/acs.chemrev.8b00031Crossref PubMed Scopus (80) Google Scholar, S. S. for the second oxidant in the catalytic of cytochrome a theoretical of the species and its Am. Chem. Soc. PubMed Scopus Google Scholar, Q. W. D. M.T. a or two active Am. Chem. Soc. PubMed Scopus Google Scholar, M. D. R. Kinetic isotope effects two electrophilic in cytochrome Am. Chem. Soc. PubMed Scopus Google Scholar), although the that reactions involve the third step of CYP51 catalysis can a ferric peroxo mechanism, we applied a crystallographic and biochemical The was on our that substrate binding a conformational in the structure of CYP51 from the protozoan T. T.Y. Wawrzak Z. S.A. Guengerich F.P. Waterman M.R. Lepesheva G.I. Binding of a physiological substrate conformational in cytochrome P450 Biol. Chem. 2018; 293 Full Text Full Text PDF PubMed Scopus Google Scholar). the was a of the that to the of the a for the activation of the CYP51 proton delivery network.

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