CYP51 from Trypanosoma cruzi
2005; Elsevier BV; Volume: 281; Issue: 6 Linguagem: Inglês
10.1074/jbc.m510317200
ISSN1083-351X
AutoresGalina I. Lepesheva, N.G. Zaitseva, W. David Nes, Wenxu Zhou, Miharu Arase, Jialin Liu, George C. Hill, Michael R. Waterman,
Tópico(s)Synthesis and Biological Evaluation
ResumoA 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 F. Yokoyama K. Lockman J. Aikenhead K. Ohkanda J. Sadilek M. Sebti S. Van Voorhis W. Hamilton A. Gelb M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15149-15153Crossref PubMed Scopus (70) Google Scholar). Sterol 14α-demethylases are members of the cytochrome P450 superfamily (CYP51), which catalyze oxidative removal of the 14α-methyl group from post-squalene sterol precursors (Fig. 1A). Even with only 22-33% amino acid identity across the biological kingdoms (18Lepesheva G.I. Waterman M.R. Mol. Cell. Endocrinol. 2004; 215: 165-170Crossref PubMed Scopus (93) Google Scholar), the orthologous enzymes from bacteria to mammals preserve strict catalytic regio- and stereospecificity and have a very limited range of substrates. Until now there were four CYP51 substrates known (Fig. 1B): lanosterol (L), 24,25-dihydrolanosterol (D), 24-methylanedehidrol-anosterol (M), and obtusifoliol (O), with no other compounds reported to be metabolized by this enzyme. Although mammalian and fungal orthologs in vitro are able to 14α-demethylate each of them (although only C4-dimethylsterols are formed in vivo, D/L and M/L, respectively), CYP51 from plants are specific toward their physiological substrate, O (19Lamb D.C. Kelly D.E. Kelly S.L. FEBS Lett. 1998; 425: 263-265Crossref PubMed Scopus (59) Google Scholar). We have found recently (20Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (68) Google Scholar) that, although the pathways leading to obtusifoliol formation in plants and Trypanosomatidae are different, the same profound substrate preference toward this C4-monomethylated sterol exists in CYP51 from Trypanosoma brucei (TB), the cause of African trypanosomiasis. In contrast, much to our surprise, as described herein, upon initiating studies of TCCYP51 as the target for direct testing of specific inhibitory effects of a broad range of potential anti-chagastic agents in vitro, this enzyme is found to strongly favor substrates having two C4-methyl groups. This is the first case within the same phylum that such profound differences in CYP51 substrate specificity have been observed. The substrate preferences of TC and TB CYP51 imply that the catalytic sequence after lanosterol formation in TC and TB is different. Although the physiological reason for this at the current level of our knowledge of the biology of the parasites remains unclear, we have found that the molecular basis of the substrate preferences of TCCYP51 is largely associated with a single amino acid in the B′ helix. Cloning of TCCYP51—Sequence data for the TCCYP51 gene were obtained from the website of The Institute of Genomic Research (www.tigr.org) using as a template the protein sequence of TBCYP51 (20Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (68) Google Scholar) for a tblastn homology search. The gene was PCR-amplified from TC genomic DNA using the FailSafe PCR Premix Selection kit (Epicenter). The upstream primer 5′-CGCCATATGTTCATTGAAGCCATTGTATTGG-3′ contained a unique NdeI cloning site (underlined) and corresponded to the TCCYP51 cDNA from 1 to 25 bp. The downstream primer 5′-CGCAAGCTTCAGTGATGGTGATGCGAGGGCAATTTCTTCTTGCG-3′ included a unique HindIII cloning site (underlined) followed by a stop codon (bold) and C-terminal 4-histidine tag (italics) and was complementary to TCCYP51 sequence from 1443 to 1423 bp. The reaction conditions were the same as described for cloning of TBCYP51 (20Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (68) Google Scholar), the PCR products were subcloned into pGEM-T Easy vector (Promega), and the correctness of the inserts confirmed by DNA sequencing. The TCCYP51 DNA and protein sequences were deposited into the NCBA data base (accession number AY856083). To obtain the expression construct, the TCCYP51 insert was excised by digestion with NdeI and HindIII (New England Biolabs) and cloned into the pCW expression vector (21Barnes H.J. Arlotto M.P. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5597-5601Crossref PubMed Scopus (536) Google Scholar). The expression plasmids were sequenced again and transformed into Escherichia coli HMS-174 cells (Novagen). Site-directed Mutagenesis—The QuikChange mutagenesis kit (Stratagene) was used to destroy the internal NdeI site at 1113 bp (silent mutation Ser371 Ser, TCA to TCT), to introduce N-terminal modifications in order to increase the P450 expression level in E. coli (Table 1) and also to mutate Ile105 (ATT) to Phe (TTT) and Thr107 (ACA) to Val (GTA).TABLE 1Impact of N- and C-terminal modification of the TCCYP51 gene on the P450 expression level and purification yieldConstructN terminusC terminusP450 expressionYield after Ni2+ columnnmol/l%1MFIEA4-HisATG-TTC-ATT-GAA-GCCCAT-CAC-CAT-CAC-TGA2MAIEA4-His5-10<15ATG-GCT-ATT-GAA-GCCCAT-CAC-CAT-CAC-TGA3MAIKA4-His250-300<20ATG-GCT-ATT-AAA-GCTCAT-CAC-CAT-CAC-TGA4MAIKA6-His250-30080-90ATG-GCT-ATT-AAA-GCTCAT-CAC-CAT-CAC-CAT-CAC-TGA Open table in a new tab Expression in E. coli and Purification—CYP51 from TB, human (HU), Candida albicans (CA), and Mycobacterium tuberculosis (MT) and TB and rat cytochrome P450 reductase were all expressed and purified as described previously (20Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (68) Google Scholar). TCCYP51 was expressed at conditions similar to the TBCYP51 ortholog and purified using three chromatographic stages, including a nickel-chelating affinity column as described for human CYP51 (22Lepesheva G.I. Virus C. Waterman M.R. Biochemistry. 2003; 42: 9091-9101Crossref PubMed Scopus (75) Google Scholar), negative chromatography on Q-Sepharose (Amersham Biosciences), and cation exchange on SP-Sepharose (Amersham Biosciences). Molecular weight and purity of the proteins were confirmed by SDS-PAGE. Preparation of Sterol Substrates and Synthesis of 14α-Amino-derivatized Substrate Analogs—O, L, D, and M were isolated from plant and fungal sources and labeled with 3H at C3 as described (20Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (68) Google Scholar). N was from the Nes steroid collection (23Venkatramesh M. Guo D. Jia Z. Nes W.D. Biochim. Biophys. Acta. 1996; 1299: 313-324Crossref PubMed Scopus (56) Google Scholar) and labeled under the same conditions as other sterols. 4,4-Dimethyl-14α-aminomethyl-cholest-7-en-3β-ol (AL7) and 4,4-dimethyl-14α-aminomethyl-cholest-8-en-3β-ol (AL8) were synthesized as shown in Scheme 1 (compounds 5 and 6, respectively). For routine monitoring of the product profile, steroids were injected into a GC column packed with 3% SE-30 from a Hewlett 5890 Packard Series II operated isothermally at 240 °C. Cholesterol was used as the reference specimen from which the retention times of unknown specimens relative to the retention of cholesterol were calculated. GC-mass spectroscopy was operated using a HP 6890 GC interfaced to a 5973 mass spectrometer at 70 eV. GC was performed using an Agilent HP-5 column (30 m × 25 μm in diameter). Film thickness was 0.25 μm, and the flow rate of He was set at 1.2 ml/min. The temperatures for the GC to mass spectroscopy interface, mass spectroscopy ion source, and quadruple were 280, 250, and 230 °C, respectively. Helium at 8 p.s.i. was used as the carrier gas. In this system cholesterol elutes at 13 min. 1H NMR spectra were obtained on a Varian Unity Inova 500 MHz spectrometer at ambient temperature in deuterochloroform with tetramethylsilane as the internal standard. Spectroscopic Measurements—Absolute and difference absorbance spectra of TCCYP51 were taken at room temperature using a Shimadzu UV-240IPC spectrophotometer. The high spin form content was estimated from the absolute spectra using the ratio (ΔA393-470/ΔA417-470) (20Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (68) Google Scholar). The Na2S2O4-reduced carbon monoxide complex (CO) difference spectra were used to measure the P450 concentration (molar extinction coefficient 91 mm-1 cm-1) to test correspondence between the total amount of hemoprotein (molar extinction coefficient 117 mm-1cm-1) and P450 complexes and to confirm the absence of conversion into the inactive P420 form. Specific heme content was estimated as the ratio between hemoprotein and total protein determined using the BSA protein assay reagent (Pierce). The abilities of rat and TB cytochrome P450 reductase to support electron transport from NADPH to TCCYP51 were compared as both the half time and efficiency of the enzymatically reduced CO-complex formation at 1 μm P450 and a P450:cytochrome P450 reductase molar ratio 1:2. Catalytic Activity—Reconstitution of TCCYP51 enzymatic activity was carried out at 2 μm P450 final concentration and a enzyme:substrate molar ratio 1:25. The final sample contained 4 μm TB cytochrome P450 reductase, 100 μm dilauroyl-α-phosphatidylcholine, 0.4 mg/ml isocitrate dehydrogenase, 25 mm sodium isocitrate, and 5 mm NADPH in 20 mm MOPS (pH 7.4), 50 mm KCl, 5 mm MgCl2, and 10% glycerol (buffer A). Activities of TB, HU, CA, Sorghum bicolor (SB), and MT CYP51 were compared in parallel experiments, only using rat cytochrome P450 reductase as the electron donor for HU, CA, and SB orthologs and the E. coli flavodoxin:flavodoxin reductase system (24Lepesheva G.I. Podust L.M. Bellamine A. Waterman M.R. J. Biol. Chem. 2001; 276: 28413-28420Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) for MTCYP51. After incubation for different times at 37 °C, the reaction was stopped by extraction of the sterols with ethyl acetate. The reaction products were analyzed by reverse-phase HPLC (Waters Corp.) equipped with β-RAM detector (INUS Systems Inc.) using a Varian C18 column (150 × 4.6 mm) and a linear gradient acetonitrile:water:methanol (4.5:1:4.5), methanol as a mobile phase at a flow rate 1.5 ml/min. Ligand Binding Assay—Ligand-induced changes in the Soret band of ferric TCCYP51 (2 μm) were monitored at room temperature in buffer A. The sterol substrates (N, O, L, D, and M) were added from 1 mm stock solution in 45% 2-hydroxypropyl-β-cyclodextrin (Cyclodextrin Technologies Development, Inc.) in the range 0.25-20 μm. Fluconazole (ICN Biomedicals, Inc.) and 14α-aminomethyl derivatives of L, AL7 and AL8, were dissolved in Me2SO (2 mm) and added in the range 0.1-20 μm. The corresponding volumes of 2-hydroxypropyl-β-cyclodextrin or Me2SO were added to the reference cuvette. Maximal spectral response per nanomole of P450 (type I, ΔA390-420 for substrate binding; type II, ΔA426-410 and ΔA426-390 for nitrogen coordination for fluconazole and amino derivatives of L, respectively) and apparent dissociation constants were determined as previously described (22Lepesheva G.I. Virus C. Waterman M.R. Biochemistry. 2003; 42: 9091-9101Crossref PubMed Scopus (75) Google Scholar) by plotting absorbance changes against the concentration of free ligand and fitting the data to a rectangular hyperbola using Sigma Plot Statistics. Inhibition of TCCYP51—The inhibitory effects of fluconazole and the two amino-substrate analogs were compared as TCCYP51 activity in the presence of increasing concentrations of the tested inhibitors (1-50 μm) and expressed as the molar ratio I/P450 at which the activity decreases 2-fold. Homology Modeling—A molecular model of TCCYP51 was constructed using MTCYP51 (1e9x) (41% structural identity), CYP3A4 (1tqn) (29% identity), and 2C9 (1nr6) (29% identity) as templates. The sequences were aligned, and the coordinates were obtained from the Swiss Institute of Bioinformatics. Coordinates for heme and fluconazole were taken from MTCYP51 (1ea1). Mutations were modeled in Swiss-PDB Viewer. The sequence of the amplified TCCYP51 gene corresponds to the preliminary DNA sequence in the TIGR data base with the exception of one, silent base pair substitution (G78A). The protein consists of 481 amino acid residues having a molecular mass of ∼55 kDa. Although the N-terminal part of the gene is not highly GC-rich (AT/GC ratio is 55/45 for the first 40 codons), the wild type TCCYP51 gene was not expressed in E. coli in the P450 form. Substitution of the second codon to alanine (GCT) (21Barnes H.J. Arlotto M.P. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5597-5601Crossref PubMed Scopus (536) Google Scholar) led to low P450 expression (Table 1). A further increase of the AT content of the 5′-end by silent mutation in codon 4 and insertion of the substitution E5K allowed us to increase the expression to 250-300 nmol of P450 per liter. Poor interaction of the four His-tagged TCCYP51 with the nickel column suggested limited availability of the TCCYP51 C terminus, perhaps due to proline 480 restricting its local flexibility. Insertion of two additional His residues into the TCCYP51 C-terminal His tag solved this problem. The yield of the electrophoretically pure P450 after the three-step purification procedure (Fig. 2A)was about 50%. TCCYP51 is purified in the low spin form (Fig. 2B) with a Soret maximum at 417 nm and equal intensities of α-(568 nm) and β-band (536 nm) absorbances, which is typical for sterol 14α-demethylases. The purified protein had a spectrophotometric index of A417/280 1.6 and a specific heme content of 16.6 nmol/mg. The carbon monoxide spectrum of Na2S2O4-reduced TCCYP51 is stable at room temperature for at least 30 min and had a maximum at 448 nm. P420 content does not exceed 5%. Both rat and TB cytochrome P450 reductase support electron transfer from NADPH to TCCYP51, with maximal efficiency 85 and 100% that of the amount of chemically reduced P450, respectively. The electron transfer, however, at least in in vitro conditions, occurred faster with the reductase from TB (Supplemental Fig. 2). The fact that TB cytochrome P450 reductase is a better electron donor for TCCYP51 correlates with our previous data on the reduction of TBCYP51 (20Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (68) Google Scholar) and implies that the two protozoan CYP51s are likely to have high similarity in the organization of the region of interaction with their electron donor partner. Preferences of TB and TC CYP51 for sterol substrates are remarkably different. Although TBCYP51 profoundly prefers O of the four known substrates of sterol 14α-demethylase (20Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (68) Google Scholar), TCCYP51 demethylates this sterol very poorly (0.06 min-1) (Table 2). Instead, it shows the highest turnover number upon 14α-demethylation of M (2.4 min-1). Metabolism of D upon the same conditions is about 30% slower (1.6 min-1). The rate of 14α-demethylation of L is only 0.57 min-1, although the initial accumulation of the 14α-carboxyaldehyde intermediate from L occurs more than 3-fold faster than the overall demethylation reaction (1.7 min-1). The tendency of TCCYP51 to form the aldehyde intermediate from L resembles that observed with TBCYP51, although the ratio between the rates of formation of the intermediate and the product from L by TBCYP51 is much higher, reaching about 80 (20Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (68) Google Scholar). Substrate binding parameters calculated from type I spectral responses of TCCYP51 (blue shift from 417 to 393 nm in the Soret band) upon sterol versus 2-hydroxypropyl-β-cyclodextrin addition, including maximal amplitude, which reflects the increase in the high spin content (hs,%), apparent dissociation constants (Kd), and their ratio (hs/Kd), suggest that the differences in the rates of sterol metabolism are connected with the ability of the enzyme to interact with the substrate. Comparison of the values calculated for M, D, and L demonstrates a role for the geometry of the hydrophobic side chain. Both M and D have a flexible side chain, but the larger side chain size caused by the additional 24-methylene group of M in this region is slightly preferable. Alternatively, a decrease of the side chain flexibility by the addition of a 24 (25Goad L.J. Holz Jr., G.G. Beach D.H. Mol. Biochem. Parasitol. 1985; 3: 257-279Crossref Scopus (58) Google Scholar)-double bond (L) further affects interaction (∼2-fold) and product formation (∼3-fold), although the rate of production of the aldehyde intermediate remains practically the same. This 2-fold reduction due to the side chain alteration is also observed upon comparison of the binding and metabolism of O and its C24-demethylated analog N (which we tested as a potential substrate for trypanosomal CYP51 because it was listed among the sterols identified in vivo in Leishmania species (25Goad L.J. Holz Jr., G.G. Beach D.H. Mol. Biochem. Parasitol. 1985; 3: 257-279Crossref Scopus (58) Google Scholar, 26Beach D.H. Goad L.J. Holz Jr., G.G. Mol. Biochem. Parasitol. 1988; 2: 149-162Crossref Scopus (101) Google Scholar)). However, the major differences in catalytic activity and binding parameters (M, D, and L versus O and N) are derived from the geometry surrounding the sterol C4 atom; both the equatorial (α) and the angular (β) methyl groups are strongly favored by TCCYP51.TABLE 2Sterol binding and catalytic activity of TCCYP51Wild typeI105FSubstrateSpectral responseTurnovercnmol of substrate/nmol of P450/min.Spectral responseTurnoverEffect (-fold)hsmaMaximal increase in the high spin content calculated from the spectral response per nmol of P450, where 0.11 optical units/nmol corresponds to 100% conversion of TCCYP51 from low to high spin form.KdbApparent dissociation constant.hsm/Kdhsm%Kdhsm/KdInteractiondhsm/Kd mutant/hsm/Kd wild type.Activity%μm%μmN10 ± 0.79.5 ± 1.41.20.03 ± 0.0154 ± 2.20.04 ± 0.011354.6 ± 0.5↑ 113↑ 150O9 ± 0.64.4 ± 0.72.00.06 ± 0.0145 ± 3.40.7 ± 0.05643.6 ± 0.3↑ 32↑ 60L21 ± 1.81.9 ± 0.3110.57 ± 0.04eThe rate of accumulation of the 14α-aldehyde intermediate is 1.7 min−1. Results are presented as the mean ± S.D. of four experiments.20 ± 2.12.3 ± 0.28.70.16 ± 0.02↓ 1.3↓ 3.6D30 ± 1.71.2 ± 0.1251.6 ± 0.123 ± 2.01.4 ± 0.2210.42 ± 0.03↓ 1.2↓ 3.8M36 ± 0.90.8 ± 0.1452.4 ± 0.135 ± 2.70.9 ± 0.1390.71 ± 0.05↓ 1.3↓ 3.4a Maximal increase in the high spin content calculated from the spectral response per nmol of P450, where 0.11 optical units/nmol corresponds to 100% conversion of TCCYP51 from low to high spin form.b Apparent dissociation constant.c nmol of substrate/nmol of P450/min.d hsm/Kd mutant/hsm/Kd wild type.e The rate of accumulation of the 14α-aldehyde intermediate is 1.7 min−1. Results are presented as the mean ± S.D. of four experiments. Open table in a new tab It is generally accepted that plant sterol 14α-demethylases differ from the rest of the CYP51 family by being specific toward their physiological substrate O (27Taton M. Rahier A. Biochem. J. 1991; 277: 483-492Crossref PubMed Scopus (66) Google Scholar, 28Kahn R.A. Bak S. Olsen C.E. Svendsen I. Moller B.L. J. Biol. Chem. 1996; 271: 32944-32950Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 29Cabello-Hurtado F. Taton M. Forthoffer N. Kahn R. Bak S. Rahier A. Werck-Reichhart D. Eur. J. Biochem. 1999; 262: 435-446Crossref PubMed Scopus (45) Google Scholar, 30Waterman M.R. Lepesheva G.I. Biochem. Biophys. Res. Commun. 2005; 338: 418-422Crossref PubMed Scopus (61) Google Scholar), although the structural basis for this preference remains unknown. We have found recently that the same strict specificity toward obtusifoliol is demonstrated by TBCYP51 (20Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (68) Google Scholar). In this study reconstitution of the reaction of 14α-demethylation of the five sterols by mammalian (HU), fungal (CA), bacterial (MT), plant (SB), and TB CYP51 (Fig. 3A) showed t
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