CYP51 from Trypanosoma cruzi

Abstract

A potential drug target for treatment of Chagas disease, sterol 14α-demethylase from Trypanosoma cruzi (TCCYP51), was found to be catalytically closely related to animal/fungi-like CYP51. Contrary to the ortholog from Trypanosoma brucei (TB), which like plant CYP51 requires C4-monomethylated sterol substrates, TCCYP51 prefers C4-dimethylsterols. Sixty-six CYP51 sequences are known from bacteria to human, their sequence homology ranging from ∼25% between phyla to ∼80% within a phylum. TC versus TB is the first example of two organisms from the same phylum, in which CYP51s (83% amino acid identity) have such profound differences in substrate specificity. Substitution of animal/fungi-like Ile105 in the B′ helix to Phe, the residue found in this position in all plant and the other six CYP51 sequences from Trypanosomatidae, dramatically alters substrate preferences of TCCYP51, converting it into a more plant-like enzyme. The rates of 14α-demethylation of obtusifoliol and its 24-demethyl analog 4α-,4α-dimethylcholesta-8,24-dien-3β-ol(norlanosterol) increase 60- and 150-fold, respectively. Turnover of the three 4,4-dimethylated sterol substrates is reduced ∼3.5-fold. These catalytic properties correlate with the sterol binding parameters, suggesting that Phe in this position provides necessary interactions with C4-monomethylated substrates, which Ile cannot. The CYP51 substrate preferences imply differences in the post-squalene portion of sterol biosynthesis in TC and TB. The phyla-specific residue can be used to predict preferred substrates of new CYP51 sequences and subsequently for the development of new artificial substrate analogs, which might serve as highly specific inhibitors able to kill human parasites. A potential drug target for treatment of Chagas disease, sterol 14α-demethylase from Trypanosoma cruzi (TCCYP51), was found to be catalytically closely related to animal/fungi-like CYP51. Contrary to the ortholog from Trypanosoma brucei (TB), which like plant CYP51 requires C4-monomethylated sterol substrates, TCCYP51 prefers C4-dimethylsterols. Sixty-six CYP51 sequences are known from bacteria to human, their sequence homology ranging from ∼25% between phyla to ∼80% within a phylum. TC versus TB is the first example of two organisms from the same phylum, in which CYP51s (83% amino acid identity) have such profound differences in substrate specificity. Substitution of animal/fungi-like Ile105 in the B′ helix to Phe, the residue found in this position in all plant and the other six CYP51 sequences from Trypanosomatidae, dramatically alters substrate preferences of TCCYP51, converting it into a more plant-like enzyme. The rates of 14α-demethylation of obtusifoliol and its 24-demethyl analog 4α-,4α-dimethylcholesta-8,24-dien-3β-ol(norlanosterol) increase 60- and 150-fold, respectively. Turnover of the three 4,4-dimethylated sterol substrates is reduced ∼3.5-fold. These catalytic properties correlate with the sterol binding parameters, suggesting that Phe in this position provides necessary interactions with C4-monomethylated substrates, which Ile cannot. The CYP51 substrate preferences imply differences in the post-squalene portion of sterol biosynthesis in TC and TB. The phyla-specific residue can be used to predict preferred substrates of new CYP51 sequences and subsequently for the development of new artificial substrate analogs, which might serve as highly specific inhibitors able to kill human parasites. Trypanosoma cruzi (TC) 2The abbreviations used are: TC, T. cruzi; TB, T. brucei; SB, S. bicolor; CA, C. albicans; MT, M. tuberculosis; HU, human; CYP, cytochrome P450 gene or protein; CYP51, sterol 14α-demethylase; L, lanosterol; D, 24, 25-dihydrolanosterol; M, 24-methylenedihydrol-anosterol; O, obtusifoliol; N, norlanosterol (4α, 14α-dimethylcholesta-8,24-dien-3β-ol); AL7, 4,4-dimethyl-14α-aminomethyl-cholest-7-en-3β-ol; AL8, 4,4-dimethyl-14α-aminomethyl-cholest-8-en-3β-ol; HPLC, high performance liquid chromatography; GC, gas chromatography; MOPS, 4-morpholinepropanesulfonic acid. 2The abbreviations used are: TC, T. cruzi; TB, T. brucei; SB, S. bicolor; CA, C. albicans; MT, M. tuberculosis; HU, human; CYP, cytochrome P450 gene or protein; CYP51, sterol 14α-demethylase; L, lanosterol; D, 24, 25-dihydrolanosterol; M, 24-methylenedihydrol-anosterol; O, obtusifoliol; N, norlanosterol (4α, 14α-dimethylcholesta-8,24-dien-3β-ol); AL7, 4,4-dimethyl-14α-aminomethyl-cholest-7-en-3β-ol; AL8, 4,4-dimethyl-14α-aminomethyl-cholest-8-en-3β-ol; HPLC, high performance liquid chromatography; GC, gas chromatography; MOPS, 4-morpholinepropanesulfonic acid. is a pathogenic protozoon, the causative agent of Chagas disease, or American trypanosomiasis, threatening lives of more than 100 million people (1El-Sayed N.M. Myler P.J. Bartholomeu D.C. et al.Science. 2005; 309: 409-415Crossref PubMed Scopus (1148) Google Scholar). The parasite has a complex life cycle, which involves obligatory passage through four stages, proliferative epimastigote and infective trypomastigote in triatomine insects as vectors, proliferative intracellular amastigotes, and infective bloodstream trypomastigotes in mammalian hosts (2Kreier T. Julius P. Parasitic Protozoa. 2nd Ed. Academic Press, Inc., San Diego, CA1992Google Scholar). The infection is transmitted among humans predominantly by insect vectors or through blood transfusion and primarily affects the heart (chagastic cardiopathy), gastrointestinal tract (megasyndromes), and nervous system (dementia) (3Morel C.M. Lazdins J. Nat. Rev. Microbiol. 2003; 1: 14-15Crossref PubMed Scopus (67) Google Scholar). Left untreated, Chagas disease is commonly fatal. Anti-parasitic therapy is highly toxic and usually ineffective in the chronic stages (1El-Sayed N.M. Myler P.J. Bartholomeu D.C. et al.Science. 2005; 309: 409-415Crossref PubMed Scopus (1148) Google Scholar, 4Coura J.R. Castro S.L. Mem. Inst. Oswaldo Cruz. 2002; 97: 3-24PubMed Google Scholar). Growing knowledge on the basic biology of TC opens new opportunities for rationally developed approaches to treatment of Chagas disease. One of the approaches is to block a key metabolic pathway in the parasite (5Urbina J.A. Curr. Opin. Infect. Dis. 2001; 6: 733-741Crossref Scopus (90) Google Scholar). Conserved in eukaryota, sterol biosynthesis is one such target pathway. Either through deoxyxylulose or more generally via the mevalonate pathway (which is likely to take place in Trypanosomatidae (6Berriman M. Ghedin E. Hertz-Fowler C. et al.Science. 2005; 309: 416-422Crossref PubMed Scopus (1327) Google Scholar)), it leads to squalene cyclization in all sterol-synthesizing organisms (www.genome.ad.jp/kegg/pathway/map/map00100.html) and then proceeds through several phyla-specific reactions of sterol core modification, including C4 and C14 demethylation and side-chain modification, resulting in production of cholesterol in animals, ergosterol in fungi, and a variety of 24 alkylated and olefinated sterols in plants and protozoa (7Nes W.R. McKean M.R. Biochemistry of Steroids and Other Iisopentenoids. University Park Press, Baltimore, MD1977: 229-270Google Scholar, 8Schaller H. Prog. Lipid Res. 2003; 42: 163-175Crossref PubMed Scopus (272) Google Scholar, 9Roberts C.W. McLeod R. Rice D.W. Ginger M. Chance M.L. Goad L.J. Mol. Biochem. Parasitol. 2003; 126: 129-142Crossref PubMed Scopus (249) Google Scholar). The sterols are essential architectural components of cellular membranes and also fulfill bioregulatory functions, e.g. serving as precursors of steroid hormones and vitamin D in mammals and modulators of growth and development in unicellular organisms (9Roberts C.W. McLeod R. Rice D.W. Ginger M. Chance M.L. Goad L.J. Mol. Biochem. Parasitol. 2003; 126: 129-142Crossref PubMed Scopus (249) Google Scholar). Although animals can accumulate cholesterol from the diet, blocking ergosterol production is lethal in fungi, making fungal sterol biosynthesis a very attractive target for treatment of human mycoses (10Kale P. Johnson L.B. Drugs Today. 2005; 41: 91-105Crossref PubMed Scopus (76) Google Scholar). In Trypanosomatidae, structural sterols are found in plasma, inner mitochondrial, and glycosomal membranes (11Catisti R. Uyemura S.A. Vercesi A.E. Lira R. Rodriguez C. Urbina J.A. Docampo R. J. Eukaryot. Microbiol. 2001; 48: 588-594Crossref PubMed Scopus (54) Google Scholar, 12Quinones W. Urbina J.A. Dubourdieu M Luis Concepcion J. Exp. Parasitol. 2004; 106: 135-149Crossref PubMed Scopus (37) Google Scholar). Depletion of sterol end products causes trypanosomal cell death as a result of membrane disruption, being especially effective in the exponentially dividing stages of the parasite life cycle (13Braga M.V. Urbina J.A. de Souza W. Int. J. Antimicrob. Agents. 2004; 1: 72-78Crossref Scopus (47) Google Scholar, 14Garzoni L.R. Caldera A. Meirelles M.N. de Castro S.L. Docampo R. Meints G.A. Oldfield E. Urbina J.A. Int. J. Antimicrob. Agents. 2004; 3: 273-285Crossref Scopus (89) Google Scholar). Among the genes encoding sterol biosynthetic enzymes in the TC genome (www.tigr.org), sterol 14α-demethylase, as a primary target of clinical antifungal azoles, is of special interest. It is well established that several imidazole and triazole derivatives are highly effective against TC, causing the parasite cell growth inhibition and increasing survival of infected animals (15Urbina J.A. Payares G. Molina J. Sanoja C. Liendo A. Lazardi K. Piras M.M. Piras R. Perez N. Wincker P. Ryley J.F. Science. 1996; 273: 969-971Crossref PubMed Scopus (176) Google Scholar, 16Araujo M.S. Martins-Filho O.A. Pereira M.E. Brener Z. J. Antimicrob. Chemother. 2000; 45: 819-824Crossref PubMed Scopus (73) Google Scholar, 17Buckner K. J. K. J. M. S. W. A. S. A. 2003; PubMed Scopus Google Scholar). are of the cytochrome P450 which of the from post-squalene sterol precursors with amino acid the M.R. Mol. 2004; PubMed Scopus Google the enzymes from bacteria to mammals catalytic and and have a very of four CYP51 substrates known and obtusifoliol with other to be by this enzyme. Although mammalian and fungal in are able to of are in and CYP51 from plants are specific their D.C. S.L. PubMed Scopus Google Scholar). have found W. M.R. 2004; PubMed Scopus Google the to obtusifoliol in plants and Trypanosomatidae are the same profound substrate this C4-monomethylated sterol in CYP51 from Trypanosoma brucei (TB), the of In to as of TCCYP51 as the target for of specific of a of potential in this is found to substrates two is the first within the same that such profound differences in CYP51 substrate have The substrate preferences of TC and TB CYP51 imply that the catalytic sequence in TC and TB is Although the for this the of knowledge of the biology of the have found that the of the substrate preferences of TCCYP51 is with a amino acid in the B′ of for the TCCYP51 gene from the of The of as a the sequence of W. M.R. 2004; PubMed Scopus Google for a homology The gene was from TC the The a and to the TCCYP51 from to The a by a and and was to TCCYP51 sequence from to The the same as for of W. M.R. 2004; PubMed Scopus Google the products into and the of the by The TCCYP51 and sequences into the the the TCCYP51 was by with and and into the M.R. S. A. PubMed Scopus Google Scholar). The and into was used to the to to in to increase the P450 in E. and also to Ile105 to Phe and to of and of the TCCYP51 gene on the P450 and in a new in E. and from TB, human and and TB and cytochrome P450 all and as W. M.R. 2004; PubMed Scopus Google Scholar). TCCYP51 was to the ortholog and three stages, including a as for human CYP51 C. M.R. 2003; 42: PubMed Scopus Google on and on and of the by of and of L, D, and M from plant and fungal and with as W. M.R. 2004; PubMed Scopus Google Scholar). was from the steroid M. Z. 1996; PubMed Scopus Google and the same as other and as in and of the into a with from a was used as the from which the of to the of cholesterol was a to a was in was and the of was The for the to and and respectively. was used as the In this system cholesterol on a in with as the and of TCCYP51 a The high was from the the W. M.R. 2004; PubMed Scopus Google Scholar). The complex used to the P450 to between the of and P450 and to the of into the was as the between and the The of and TB cytochrome P450 to from to TCCYP51 as the and of the reduced P450 and a P450 of TCCYP51 was P450 and a The TB cytochrome P450 100 and in and of TB, HU, CA, and CYP51 in cytochrome P450 as the for HU, CA, and and the E. system A. M.R. J. 2001; PubMed Scopus Google for for the was by of the sterols with The products by with a and a as a a in the of TCCYP51 in A. The sterol substrates O, L, D, and from in in the and derivatives of L, and AL8, in and in the The of or to the of P450 for substrate and for for and amino derivatives of L, and as C. M.R. 2003; 42: PubMed Scopus Google by against the of and the to a of of and the two as TCCYP51 in the of increasing of the inhibitors and as the which the of TCCYP51 was structural and identity) as The sequences and the from the of for and from in The sequence of the TCCYP51 gene to the sequence in the with the of The of amino acid a of Although the of the gene is highly is for the first the TCCYP51 gene was in E. in the P450 Substitution of the to M.R. S. A. PubMed Scopus Google to P450 A increase of the of the by in and of the to increase the to of P450 of the four TCCYP51 with the of the TCCYP51 to its of two into the TCCYP51 this The of the P450 the TCCYP51 is in the with a and of and which is for sterol The a of and a specific of The of TCCYP51 is for and a and TB cytochrome P450 from to TCCYP51, with and that of the of reduced respectively. The in in with the from TB The that TB cytochrome P450 is a for TCCYP51 with on the of W. M.R. 2004; PubMed Scopus Google and that the two CYP51s are likely to have high in the of the of with their of TB and TC CYP51 for sterol substrates are Although prefers of the four known substrates of sterol 14α-demethylase W. M.R. 2004; PubMed Scopus Google TCCYP51 this sterol very it the 14α-demethylation of M of D the same is The of 14α-demethylation of is the of the from more than than the demethylation The of TCCYP51 to the from that with the between the rates of of the and the from by is W. M.R. 2004; PubMed Scopus Google Scholar). binding from of TCCYP51 from to in the sterol versus including which the increase in the high and their that the differences in the rates of sterol are with the of the to with the of the for M, D, and a for the of the M and D have a the by the of M in this is a of the by the of a 24 L.J. Mol. Biochem. Parasitol. 3: Scopus Google affects and the of production of the the to the is also of the binding and of and its analog (which as a potential substrate for trypanosomal CYP51 it was among the sterols in in L.J. Mol. Biochem. Parasitol. 3: Scopus Google Scholar, Goad L.J. Mol. Biochem. Parasitol. Scopus Google the differences in catalytic and binding D, and versus and are from the the sterol C4 the and the are by binding and catalytic of of of increase in the high from the of to of TCCYP51 from to high of of the is are as the of four increase in the high from the of to of TCCYP51 from to high of of The of of the is are as the of four in a new It is generally that plant sterol from the of the CYP51 by being specific their substrate M. A. Biochem. J. PubMed Scopus Google Scholar, S. J. 1996; PubMed Scopus (47) Google Scholar, M. N. R. S. A. J. Biochem. PubMed Scopus Google Scholar, M.R. Biochem. Res. 2005; PubMed Scopus Google the structural for this have found that the same obtusifoliol is by W. M.R. 2004; PubMed Scopus Google Scholar). In this of the of 14α-demethylation of the sterols by mammalian fungal plant and TB CYP51 that all the including plant and TB can also N. that it is the for the of the the sterol C4 the side-chain that plant and catalytically from the other The other sterol 14α-demethylation of all the the rates of their are TC, CA, and CYP51 C4 Although TCCYP51 is the example among which able to and C4 substrates and among the TC ortholog as a for two the substrates of mammalian and fungal CYP51 in M, are and the and sterol to preferences their substrates, D and M, D.C. S.L. PubMed Scopus Google Scholar, Biochem. Res. PubMed Scopus Google Scholar). the sequence homology between the CYP51s the differences on their substrate the from TB and TC to plant-like and of sterol 14α-demethylase, respectively. of on and of trypanosomal CYP51s with from other phyla two amino acid which are animal/fungi-like in TC and in TB CYP51 and in the B′ helix The B′ helix the of cytochrome P450 substrate J. Google Scholar). in the substrate in substrate have for M. PubMed Scopus Google Scholar, C. and 2nd Ed. Google Scholar). In the of TCCYP51, the of is to the into the portion of the substrate binding the of to the of the B′ helix of in the is to that of the residue in the of with a steroid The C4 to suggesting that of substrates in the plant CYP51 is the same as the of in the then Phe in this position might binding of with their M.R. 2004; PubMed Scopus (76) Google Scholar). Although the TCCYP51 provides for the to the of was into amino acid between the and TC to a of a of two the versus in plants and TB and all sterol Substitution as a or in the dramatically alters substrate preferences of The of or a to substrate the high of to and the of in the TCCYP51 the same is than in on the the of the with and in the and of sterols of Ile105 to Phe, of the to the of of M, D, or to 24, and to high and are to that of the it is likely that in TCCYP51 as a result of differences in the or of the substrate in to the of in Phe position with the of the substrates their it interactions with the substrates the which Ile cannot. It is the increase in is with than a in the that it might be with the of the which the for the with the of Phe more In this it be that the position of the of provides for mammalian and fungal CYP51 to with such a in TCCYP51 has binding of C4-monomethylated substrates to the profound increase in their of and more than of is by within the the TCCYP51 a of from The of in to the and is a the of the with is than the of the with the preferred substrate Although from the in to L, D, and M the of their 14α-demethylation of the suggesting that the of the sterols is in the with the residue position that of Ile105 to Phe in TCCYP51 the of binding of sterols in the of the the three of of on the of the on the of TCCYP51 with to this phyla-specific residue also to the in of this antifungal drug for and W. M.R. 2004; PubMed Scopus Google especially into that in the of to Ile105 in TC, in TB, and in CYP51 the the TCCYP51 has to to and the for the was found to be a with are in which alters substrate preferences of TCCYP51, has on its with of sterol derivatives is well known de and 2nd Ed. Google and has and are the used clinical antifungal (10Kale P. Johnson L.B. Drugs Today. 2005; 41: 91-105Crossref PubMed Scopus (76) Google Scholar). binding of TCCYP51 to of a of derivatives as potential very especially into the of on TC in (15Urbina J.A. Payares G. Molina J. Sanoja C. Liendo A. Lazardi K. Piras M.M. Piras R. Perez N. Wincker P. Ryley J.F. Science. 1996; 273: 969-971Crossref PubMed Scopus (176) Google Scholar, 16Araujo M.S. Martins-Filho O.A. Pereira M.E. Brener Z. J. Antimicrob. Chemother. 2000; 45: 819-824Crossref PubMed Scopus (73) Google Scholar, 17Buckner K. J. K. J. M. S. W. A. S. A. 2003; PubMed Scopus Google Scholar). antifungal are known to other human W. T. H. 2002; PubMed Scopus Google and generally have life in K. J. K. J. M. S. W. A. S. A. 2003; PubMed Scopus Google Scholar, S.A. 2004; PubMed Scopus Google Scholar). to CYP51 substrate to cholesterol biosynthesis in humans have that derivatives of are effective as M. T. N. N. J. Biochem. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Rev. Biochem. Mol. PubMed Scopus Google Scholar, S. Johnson P. Scopus Google Scholar). of the of to have a in as CYP51 inhibitors and as of A Rev. Biochem. Mol. PubMed Scopus Google Scholar, N. M. PubMed Scopus Google Scholar). have binding and inhibition of TCCYP51 with two substrate analogs, derivatives of L, and These have found effective against a on has J. R. J. Scopus Google Scholar). In TCCYP51 sterols with of and for and AL8, The in the might imply that is more for of the amino to the M is the it with the amino derivatives for the causing in of the from the of the the of on TCCYP51 is the A in the of 14α-demethylation of M is and of and the of M in the is the amino derivatives of the of TCCYP51, the is than the inhibition with the clinical antifungal into that the two sterols a on M. R. W. against growth of and W. and are potential for as in substrate preferences of the TB and TC CYP51s imply that the two have developed differences in the sequence of reactions in the post-squalene portion of their sterol biosynthesis In of the preferred CYP51 substrate a of L, which is the first in the pathway W. M.R. 2004; PubMed Scopus Google Scholar, Antimicrob. Chemother. 2001; 45: PubMed Scopus Google Scholar). Although in TC has to be to M, in TB one is is used as a substrate of CYP51, in in accumulate the of this in the of the is W. M.R. 2004; PubMed Scopus Google Scholar). In the of is known to of blocking the of cholesterol biosynthesis Rev. Biochem. Mol. PubMed Scopus Google Scholar, D.C. R. M. E. J. A. S. G. Biochem. PubMed Scopus Google Scholar, J. PubMed Google Scholar). Trypanosomatidae might the to the sterol production and the pathway to the of The of CYP51 in is by the that in the of Chagas disease is more than several inhibitors of in of the parasite the are Antimicrob. Chemother. 2001; 45: PubMed Scopus Google Scholar). It is that in the of TB, which the of of (6Berriman M. Ghedin E. Hertz-Fowler C. et al.Science. 2005; 309: 416-422Crossref PubMed Scopus (1327) Google including a high of A. J. A. 2001; PubMed Scopus Google the CYP51 of sterol biosynthesis from to precursors to structural sterol is more of TC in the human the of for exponentially dividing is this of the life cycle the sterol biosynthetic pathway in the parasite is and A. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar). a TC the sterol of the epimastigote and stages (15Urbina J.A. Payares G. Molina J. Sanoja C. Liendo A. Lazardi K. Piras M.M. Piras R. Perez N. Wincker P. Ryley J.F. Science. 1996; 273: 969-971Crossref PubMed Scopus (176) Google Scholar). all the products in are and the of are or Depletion of sterols with TC membranes also affects (13Braga M.V. Urbina J.A. de Souza W. Int. J. Antimicrob. Agents. 2004; 1: 72-78Crossref Scopus (47) Google Scholar, C. M. C. S.L. de K. Urbina J. de Souza W. Antimicrob. Chemother. 2004; 48: PubMed Scopus Google Scholar). The is in with the that sterols in Trypanosomatidae might be essential as of their cell cycle and development (9Roberts C.W. McLeod R. Rice D.W. Ginger M. Chance M.L. Goad L.J. Mol. Biochem. Parasitol. 2003; 126: 129-142Crossref PubMed Scopus (249) Google Scholar). Although the of of such bioregulatory sterols has it is that the substrate of TCCYP51 M might be to the of the pathway that leads to their amino acid in the B′ helix alters substrate of TCCYP51, converting it into a more enzyme. the of the TCCYP51 substrate preferences in to of is with the of Ile of Phe position in to Phe, the of the Ile is for of with or N, suggesting that in the products of the TCCYP51 are the for this to be the gene from in the of Trypanosomatidae N.M. Myler P.J. G. et al.Science. 2005; 309: PubMed Scopus Google the has from and the has in the development of the TCCYP51 substrate the of TCCYP51 to the being a result of The that the the to and that the of the TCCYP51 substrate binding has also or that TCCYP51 has other from in the TB ortholog that A that CYP51 from as the preferred the to M. A. 2005; PubMed Scopus Google provides for the The essential of three in the B′ helix in the binding and of the sterol substrates was established by of and CYP51 and in in C. M.R. 2003; 42: PubMed Scopus Google Scholar). These to the of TCCYP51 that the in the substrate a P450 as a sterol 14α-demethylase, Phe or animal/fungi-like in TC is in can be used to predict substrate preferences of new CYP51 the six other CYP51 sequences from Trypanosomatidae Phe in this it is likely that in to TC in the brucei and TB and and in the and the post-squalene of the sterol biosynthetic pathway through the 14α-demethylation of Although in the system in prefers O, on the catalytic properties of from TB as the preferred substrate of the G. M. R. and W. for that in the of this might predominantly 14α-demethylation of into that this sterol was also in in their treatment with antifungal L.J. Mol. Biochem. Parasitol. 3: Scopus Google Scholar, Goad L.J. Mol. Biochem. Parasitol. Scopus Google and in TB with sterol well be the preferred substrate of CYP51 in several Trypanosomatidae is the substrate of sterol for TC and of for CYP51. with