The Mechanism of the Acyl-Carbon Bond Cleavage Reaction Catalyzed by Recombinant Sterol 14α-Demethylase of Candida albicans (Other Names Are: Lanosterol 14α-Demethylase, P-45014DM, and CYP51)
1996; Elsevier BV; Volume: 271; Issue: 21 Linguagem: Inglês
10.1074/jbc.271.21.12445
ISSN1083-351X
AutoresAkbar Z. Shyadehi, David C. Lamb, Steven L. Kelly, Diane Kelly, Wolf‐Hagen Schunck, J. Neville Wright, David L. Corina, Muhammad Akhtar,
Tópico(s)Steroid Chemistry and Biochemistry
ResumoThe Candida albicans sterol 14α-demethylase gene (P-45014DM, CYP51) was transferred to the yeast plasmid YEp51 placing it under the control of the GAL10 promoter. The resulting construct (YEp51:CYP51) when transformed into the yeast strain GRF18 gave a clone producing 1.5 μmol of P-450/liter of culture, the microsomal fraction of which contained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursors for the 14α-demethylase, 3β-hydroxylanost-7-en-32-al and 3β-hydroxylanost-7-en-32-ol, variously labeled with 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-16O]- and [32-2H,32-18O]3β-hydroxylanost-7-en-32-al with the recombinant 14α-demethylase was performed under 16O2 or 18O2 and the released formic acid analyzed by mass spectrometry. The results showed that in the acyl-carbon bond cleavage step (i.e. the deformylation process) the original carbonyl oxygen at C-32 of the precursor is retained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously been observed for the acyl-carbon cleavage steps catalyzed by aromatase (P-450arom) and 17α-hydroxylase-17,20-lyase (P-45017α,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14α-demethylase is considered. The Candida albicans sterol 14α-demethylase gene (P-45014DM, CYP51) was transferred to the yeast plasmid YEp51 placing it under the control of the GAL10 promoter. The resulting construct (YEp51:CYP51) when transformed into the yeast strain GRF18 gave a clone producing 1.5 μmol of P-450/liter of culture, the microsomal fraction of which contained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursors for the 14α-demethylase, 3β-hydroxylanost-7-en-32-al and 3β-hydroxylanost-7-en-32-ol, variously labeled with 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-16O]- and [32-2H,32-18O]3β-hydroxylanost-7-en-32-al with the recombinant 14α-demethylase was performed under 16O2 or 18O2 and the released formic acid analyzed by mass spectrometry. The results showed that in the acyl-carbon bond cleavage step (i.e. the deformylation process) the original carbonyl oxygen at C-32 of the precursor is retained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously been observed for the acyl-carbon cleavage steps catalyzed by aromatase (P-450arom) and 17α-hydroxylase-17,20-lyase (P-45017α,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14α-demethylase is considered. INTRODUCTIONOur earlier studies on the removal of C-19 of androgens in the formation of estrogens (1.Skinner S.J.M. Akhtar M. Biochem. J. 1969; 114: 75-81Crossref PubMed Scopus (76) Google Scholar, 2.Akhtar M. Calder M.R. Corina D.L. Wright J.N. Biochem. J. 1982; 201: 569-580Crossref PubMed Scopus (272) Google Scholar, 3.Stevenson, D. E., Wright, J. N., Akhtar, M. (1988) J. Chem. Soc. Perkin Trans. I 2043–2052Google Scholar) and of the 14α-methyl group of lanosterol during sterol biosynthesis (Fig. SI, Conversion 1→4) (4.Akhtar M. Alexander K. Boar R.B. McGhie J.F. Barton D.H.R. Biochem. J. 1978; 169: 449-463Crossref PubMed Scopus (60) Google Scholar) raised the possibility that these seemingly unrelated conversions may occur by closely related mechanisms involving three steps as shown in Fig. R1.Figure R1:Reaction 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)These studies also indicated that in each case the same catalyst was responsible for all three reactions, and this feature was firmly established through genetic studies and purification to homogeneity of the two enzymes, aromatase (P-450arom) (5.Corbin C.J. Graham-Lorence S. McPhaul M. Mason J.I. Mendelson C.R. Simpson E.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8948-8952Crossref PubMed Scopus (295) Google Scholar, 6.Tan L. Muto N. Eur. J. Biochem. 1986; 156: 243-250Crossref PubMed Scopus (70) Google Scholar) and lanosterol 14α-demethylase (P-45014DM)(7.Kalb V.F. Woods C.W. Turi T.G. Dey C.R. Sutter T.R. Loper J.C. DNA (N. Y.). 1987; 6: 529-537Crossref PubMed Scopus (160) Google Scholar, 8.Aoyama Y. Yoshida Y. Sonoda Y. Sato Y. J. Biol. Chem. 1987; 262 (and references therein): 1239-1243Abstract Full Text PDF PubMed Google Scholar). The third step in estrogen biosynthesis has aroused much interest (9.Akhtar M. Njar V.C. Wright J.N. J. Steroid Biochem. Mol. Biol. 1993; 44: 375-387Crossref PubMed Scopus (139) Google Scholar, 10.Oh S.S. Robinson C.H. J. Steroid Biochem. Mol. Biol. 1993; 44: 389-397Crossref PubMed Scopus (74) Google Scholar) and the current view of the mechanism is influenced by our 18O labeling experiments(2.Akhtar M. Calder M.R. Corina D.L. Wright J.N. Biochem. J. 1982; 201: 569-580Crossref PubMed Scopus (272) Google Scholar, 3.Stevenson, D. E., Wright, J. N., Akhtar, M. (1988) J. Chem. Soc. Perkin Trans. I 2043–2052Google Scholar), which highlighted the novel nature of the process, leading to the proposal that the reaction involves an acyl-carbon cleavage represented by Fig. R2(9.Akhtar M. Njar V.C. Wright J.N. J. Steroid Biochem. Mol. Biol. 1993; 44: 375-387Crossref PubMed Scopus (139) Google Scholar).Figure R2:Reaction 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although all the experimental findings available to date on the C-C bond cleavage step in 14α-demethylation, for example the requirement for NADPH plus O2 for the reaction and release of the C1 unit as formate, could be explained (3.Stevenson, D. E., Wright, J. N., Akhtar, M. (1988) J. Chem. Soc. Perkin Trans. I 2043–2052Google Scholar, 9.Akhtar M. Njar V.C. Wright J.N. J. Steroid Biochem. Mol. Biol. 1993; 44: 375-387Crossref PubMed Scopus (139) Google Scholar) by the reaction of Fig. R2, the direct scrutiny of the hypothesis has not been possible hitherto due to the unavailability both of appropriately labeled 18O substrates and an enzyme preparation that produced sufficient formic acid for accurate 18O isotope analysis.The present paper describes a satisfactory resolution of these difficulties and reports on the status of oxygen during the C-C bond cleavage step catalyzed by lanosterol 14α-demethylase (3→4).EXPERIMENTAL PROCEDURESMaterialsIsotopically enriched 18O2 (97%) admixed with 2 volumes of argon was obtained from Isogas Limited, Croydon, Surrey and H218O was from MSD Isotopes, Montreal, Canada. Dry redistilled solvents were used, and the petroleum ether used was that with a boiling range of 60-80°C. Diazotoluene was prepared from N-benzyl-N-nitrosotoluene-4-sulfonamide (11.Corina D.L. Dunstan P.M. Anal. Biochem. 1973; 53: 571-578Crossref PubMed Scopus (13) Google Scholar). The phrase "in the usual manner" indicates that the reaction mixture was poured into water, the product extracted with ethyl acetate, the combined organic extracts washed with water, dried over anhydrous sodium sulfate, and the solvent removed under reduced pressure. All the intermediates used for the synthesis of 3β-acetoxylanost-7-en-32-onitrile (5) gave expected melting points, RF values, IR spectra, as well as mass spectrometric data.Gas Chromatography-Mass SpectrometryIsotopic distributions in the labeled substrates were determined by direct introduction probe mass spectrometric analyses of either the underivatized materials or their trimethylsilyl derivatives and are corrected for 13C natural abundance. All the mass spectra were recorded in the electron impact positive ion mode.The analysis of benzyl formate, prepared from the enzymatically produced formic acid, was performed by gas chromatography-mass spectrometry using a Hewlett-Packard 5890/VG TS-250 and a 30 m × 0.32 mm inner diameter column of DB17 with splitless injection(12.Akhtar, M., Corina, D. L., Miller, S. L., Shyadehi, A. Z., Wright, J. N. (1994) J. Chem. Soc. Perkin Trans. I, 263–267Google Scholar).The experimentally determined value for 13C natural abundance (12.4%) for benzyl formate was used to correct all the peaks between m/z 137-141, due to other isotopomers. The distribution of the isotopomers in benzyl formate was measured by comparison of the normalized ion signal areas determined by selected ion recording and corroborated by recording the full spectrum.3β-Hydroxylanost-7-en-32-al (6a)A commercially available mixture of lanost-8-en-3β-ol and lanost-8,24-dien-3β-ol (approx. 1:1) was acetylated using pyridine and acetic anhydride, and the product was recrystallized from acetone. The 24-double bond was reduced by subjecting the above mixture (59.5 g) in ethyl acetate (1 liter) and acetic acid (0.7 liter) to catalytic hydrogenation over platinum oxide (2.0 g) at room temperature and the product (54 g) recrystallized from acetone.Using the methods of Barton et al.(13.Barton, D. H. R., McGhie, J. F., Batten, P. L. (1970) J. Chem. Soc. Sect. C Org. Chem. 1033–1042Google Scholar), lanost-8-en-3β-ol acetate was converted to 3β-hydroxylanost-7-one from which was prepared 3β-acetoxylanost-7-en-32-onitrile (5) by the procedure described by Batten et al.(14.Batten, P. L., Bentley, T. J., Boar, R. B., Draper, R. W., McGhie, J. F., Barton, D. H. R. (1972) J. Chem. Soc. Perkin Trans. I 739–748Google Scholar). The nitrile (5) was reduced to 3β-hydroxylanost-7-en-32-al (6a) either with lithium aluminum hydride (14.Batten, P. L., Bentley, T. J., Boar, R. B., Draper, R. W., McGhie, J. F., Barton, D. H. R. (1972) J. Chem. Soc. Perkin Trans. I 739–748Google Scholar) or with diisobutylaluminum hydride as outlined below.3β-Acetoxylanost-7-en-32-onitrile (5) (800 mg, 1.66 mmol) in dry tetrahydrofuran (25 ml) was cooled in ice and 0.5 N diisobutylaluminum hydride in toluene (20 ml) was added slowly with stirring. The solution was allowed to stand at room temperature for 0.5 h, after which time it was poured into 10% aqueous hydrochloric acid (100 ml). The product was extracted in the usual manner, applied to a silica gel column (3 × 45 cm), and eluted with petroleum ether containing increasing amounts of ethyl acetate (up to 10%). The solvent was removed in vacuo. The resulting 3β-hydroxylanost-7-en-32-al (6a) (250 mg, 0.57 mmol) was recrystallized from methanol, m.p. 129-132°C (literature 128-130°C)(13.Barton, D. H. R., McGhie, J. F., Batten, P. L. (1970) J. Chem. Soc. Sect. C Org. Chem. 1033–1042Google Scholar), RF, 0.5 (ethyl acetate/petroleum ether, 3:7); IR (Nujol) 3540-3150 and 1720 cm–1. Mass spectrum of its trimethylsilyl derivative gave a molecular ion at m/z 514.[32-2H]3β-Hydroxylanost-7-en-32-al (6b)A procedure similar to that described immediately above was employed except that diisobutylaluminum deuteride was used for the reduction of the 3β-acetoxylanost-7-en-32-onitrile. The trimethylsilyl derivative of the product gave m/z (indicated by italics) followed by "composition; percent distribution" (indicated inside the parentheses): 515 (D1; 88%) and 514 (D1; 12%).[32-2H,32-18O]3β-Hydroxylanost-7-en-32-al (6c)[32-2H]3β-hydroxylanost-7-en-32-al (6b; 152 mg, 0.34 mmol) was dissolved in dry tetrahydrofuran (2.5 ml) containing anhydrous hydrogen chloride (25 μmol). 18O-water (310 μl) was added, and the mixture was allowed to stand at room temperature for 5.5 days, after which it was evaporated to dryness in vacuo. The residue was recrystallized from petroleum ether to give [32-2H,32-18O]3β-hydr oxylanost-7-en-32-al (6c; 112 mg, 0.25 mmol). The trimethylsilyl derivative of the latter gave m/z (indicated by italics) followed by "composition; percent distribution" (indicated inside the parentheses) 517 (D1, 18O; 76%), 516 (18O; 6%), 515 (D1; 8%), and 514 (D1; 9%).[32-2H]3β-Hydroxylanost-7-en-32-al(6e)3β-Hydroxylanost-7-en-32-al (6a) was converted to the corresponding 3β-acetoxy-compound (6a; R, CH2CO) with acetic anhydride and pyridine. The 32-carbonyl group of the resulting acetoxy compound was then reduced with sodium borotritide in the same manner as that described for the preparation of the tritium-labeled compound (7d), see below. The resulting [32-2H]3β-acetoxylanost-7-en-32-ol (7e) (40 mg, 0.081 mmol) was dissolved in acetone (10 ml) and Jones reagent (27.5 μl; 300 μl oxidizes 1 mmol of OH) was added, and the mixture was swirled for 2 min followed by extraction in the usual manner. The ensuing residue was dissolved in tetrahydrofuran (5 ml) to which was added a solution (5 ml) of 5% potassium hydroxide in methanol, the mixture left at room temperature overnight, extracted as usual, and the residue recrystallized from methanol giving [32-2H]3β-hydroxylanost-7-en-32-al (23 mg, 0.052 mmol; 15.5 μCi/μmol).3β-Hydroxylanost-7-en-32-ol (7a)To 3β-hydroxylanost-7-en-32-al (6 a; 84 mg, 019 mmol) dissolved in methanol (6 ml) and tetrahydrofuran (3 ml) was added sodium borohydride (25 mg, 0.66 mmol), and the solution was allowed to stand at room temperature for 9.5 h. The product was isolated in the usual manner and chromatographed on a silica gel column (2 × 38 cm) with petroleum ether containing increasing amounts of ethyl acetate (up to 20%) to give after crystallization from diethyl ether-petroleum ether 3β-hydroxylanost-7-en-32-ol (70 mg, 0.16 mmol), m.p. 200-203°C, RF 0.62 (ethyl acetate-petroleum ether, 1:1) and its mass spectrum gave a molecular ion at m/z 444.[32-2H2]3β-Hydroxylanost-7-en-32-ol (7b)This compound was prepared from [32-2H]3β-hydroxylanost-7-en-32-al (6b) as for the unlabeled compound (7a), but utilizing sodium borodeuteride. The recrystallized product had m/z (indicated by italics) followed by "composition; percent distribution" (indicated inside the parentheses) 446 (D2; 78%), 445 (D1; 13%), and 444 (D1; 9%).[32-2H2;32-18O]3β-Hydroxylanost-7-en-32-ol(7c)The title compound was obtained from [32-2H,32-18O]3β-hydroxylanost-7-en-3-al (6c) by reduction with sodium borodeuteride, as described above. The recrystallized product had m/z (italics), [composition; % distribution] 448[D2,18O; 38%], 447[D1,18O; 13%], 448[D2; 36%], 445[D1; 1%] and 444[D1; 12%].[32-2H]3β-Hydroxylanost-7-en-32-ol (7d)A solution of 3β-hydroxylanost-7-en-32-al (6a, 90 mg) in methanol (6 ml) and tetrahydrofuran (3 ml) was first treated with sodium borotritide (2 mg; 3-5 mCi) for 0.5 h and then unlabeled sodium borohydride (25 mg) for another 0.5 h. The reaction mixture was worked up, in the usual manner, to give 7d (14.6 μCi/μmol).Preparation of Microsomes from Pig and Rat LiverPig or rat livers were cut up into small pieces and suspended in approximately 2.5 times their volume of 100 mM potassium phosphate (also containing 2 mM glutathione, 1 mM EDTA, 4 mM magnesium chloride, 0.25 M sucrose, 0.25 mM phenylmethylsulfonyl fluoride, pH 7.4) and homogenized. The homogenate was centrifuged at 10,000 × g for 30 min. The supernatant was subsequently spun at 10,5000 × g for 1.5 h twice. The resulting microsomal pellet was resuspended in 0.1 M potassium phosphate buffer (1 mM glutathione, 0.1 mM EDTA, pH 7.4) to give a final concentration of 40-60 mg ml–1 protein.Recombinant DNA ManipulationsOur previous studies have employed a yeast expression system to express the Candida albicans CYP51 using the Saccharomyces cerevisiae phosphoglycerate kinase promotor in vector pW91P(15.Kelly S.L. Lamb D.C. Corran A.J. Baldwin B.C. Kelly D.E. Biochem. Biophys. Res. Commun. 1995; 207: 910-915Crossref PubMed Scopus (222) Google Scholar). Higher level expression was achieved by recombinant PCR ( (1)The abbreviation used is: PCRpolymerase chain reaction.) to allow transfer of CYP51 to YEp51 on a SalI/HindIII fragment and expression from the GAL10 promotor (Fig. 1). The following oligonucleotides were used as outside primers: 1, 5′-AAACTCGACAATATGGCTATTGTTGAAACTG-3′ annealing to positions 1-21 of the C. albicans CYP51(16.Lai M.H. Kirsch D.R. Nucleic Acids Res. 1989; 17: 804Crossref PubMed Scopus (102) Google Scholar) and containing a 5′-added SalI site prior to the initiating methionine and 2, 5′-TGGCATATGCATTCTGAGAGTTTCCTT-3′ annealing to the 3′ end at position 1098-1125 of the CYP51 and containing the endogenous NsiI site present in the gene.Figure 1Strategy for the cloning of the modified CYP51 gene of C. albicans. PCR mutagenesis to change the triplet at position 263 from CTG to TCT was performed using the four primers as described under "Experimental Procedures." The SalI-NsiI fragment coding for the N terminus of the protein and containing the mutation was then ligated to the C terminus encoding NsiI-HindIII fragment from pW91P, and the modified gene was inserted into SalI-HindIII cut YEp51.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Recombinant PCR was used to replace the triplet 263 (CTG) with one encoding serine (TCT) in the S. cerevisiae host. Inside primers used in the PCR mutagenesis were: 1, 5′-AAAGAAATTAAATCTAGAAGAGAA-3′ and 2, 5′ ACGTTCTCTTCTAGATTT AATTTCTTT-3′. In a first step two separate PCR reactions were performed using outside primer 1/inside primer 2 and inside primer 1/outside primer 2, respectively. The partially overlapping DNA fragments obtained were purified, mixed, and recombined in a subsequent PCR step using outside primers 1 and 2. PCR reactions were performed on a Perkin-Elmer DNA thermal cycler; conditions consisted of an initial 5 cycles of 1-min denaturation at 94°C, an annealing step for 4 min at 48°C, and an extension step for 3 min at 70°C, followed by 25 cycles of a denaturation step for 1 min at 94°C, an annealing step for 2 min at 55°C, and an extension step for 3 min at 72°C. PCR was undertaken using Pfu polymerase (Promega). Introduction of the mutation and maintenance of the authentic sequence was corroborated by DNA sequencing using Sequenase 2 (Amersham Corp.) after cloning the mutant SalI/NsiI fragment into YEp51 with ligation to the NsiI/HindIII fragment containing the C terminus and terminator regions of C. albicans CYP51. The restored CYP51 fragment was cloned directly into SalI/HindIII digested vector. All restriction enzymes and T4 DNA ligase were obtained from Promega and the recommended conditions for use were applied.Strains and TransformationsEscherichia coli strain DH5α was used for bacterial transformation and plasmid preparation. Yeast transformation to leucine prototrophy used the strain GRF18 (Matα leu2-3,2-112 his3-11, 3-15).Growth of Yeast for Heterologous ExpressionYeast transformants were grown at 28°C, 250 rpm with 250 ml of culture in 500-ml flasks. The medium used consisted of Difco yeast nitrogen base without amino acids supplemented with 100 mg/liter histidine and 2% (w/v) glucose as initial carbon source. Heterologous expression was induced when the glucose was exhausted at a cell density of approximately 108 cells/ml. The culture was left a further 4 h before the concentration of galactose was raised to 3% (w/v). After 20-h induction cells were harvested by centrifugation, resuspended in buffer containing 0.4 M sorbitol, 50 mM Tris-HCl, pH 7.4, and broken using a Braun disintegrator (Braun GmbH, Mesungen, Germany) with four bursts of 30 s together with cooling from liquid carbon dioxide. Cell debris was removed by centrifugation at 1500 × g for 5 min using a bench centrifuge. The resulting supernatant was centrifuged twice at 10,000 × g for 20 min to remove mitochondria and then at 100,000 × g for 90 min to yield the microsomal pellet. This was resuspended using a Potter-Elvehjeim glass homogenizer at about 10 mg of protein/ml in the same buffer described above. P-450 concentration by reduced carbon monoxide difference spectroscopy was measured according to (17.Omura T. Sato R. J. Biol. Chem. 1964; 239: 2370-2378Abstract Full Text PDF PubMed Google Scholar) and protein using a Sigma BCA kit.Determination of Sterol 14α-Demethylase Activity of the MicrosomesA solution of NADP+ (2 mg), glucose 6-phosphate (5 mg), and glucose-6-phosphate dehydrogenase (3 units) in 100 mM potassium phosphate buffer containing 0.1 mM EDTA, 1 mM glutathione, and 20% v/v glycerol (0.5 ml, pH 7.4) was incubated at 37°C for 20 min. To this was added microsomal protein (approximately 10 mg) and the volume made up to 1 ml with the above buffer. Following the addition of the 32-tritiated substrate (52 μg, 1.62 μCi in 10 μl of dimethylformamide), aliquots (0.1 ml) were removed (at intervals of 0, 5, 10, 30, and 60 min) and added to a mixture of dichloromethane (0.5 ml) and water (0.5 ml). The mixtures were immediately shaken and then centrifuged. The organic layer was discarded and further dichloromethane (2 × 0.5 ml) was added and the above procedure repeated. To the resulting aqueous phase was added charcoal, the suspension shaken, left at 4°C for ~1 h, and finally centrifuged to remove the charcoal. The radioactivity of the aqueous layer was measured by liquid scintillation counting.Large Scale Incubation, under Air, for the Isolation of C-32 as FormateA solution of NADP+ (5 mg), glucose 6-phosphate (10 mg), and glucose-6-phosphate dehydrogenase (5 units) in 100 mM potassium phosphate buffer containing 0.1 mM EDTA, 1 mM glutathione, and 20% glycerol (3.2 ml, pH 7.4) was incubated at 37°C for 20 min. To the incubation mixture was then added yeast microsomes (20 mg of protein in 0.8 ml of the buffer) and 0.4 mg of appropriately labeled 3β-hydroxylanost-7-en-32-al (6) or 3β-hydroxylanost-7-en-32-ol (7), admixed with tracer amounts of the 32-tritiated derivative (2.25 μCi), in dimethylformamide (40 μl). The mixture was shaken in air at 37°C for 50 min, and following acidification with 10% phosphoric acid (0.4 ml) the volatile fraction was collected by freeze-drying. The volatile fraction containing the biosynthetic formic acid was neutralized with 0.4 M sodium hydroxide (30 μl) and the solution again subjected to freeze-drying. The residue containing sodium formate was dissolved in water (3 × 100 μl) and transferred to a small vial. After the removal of water by freeze-drying, the residue containing 0.05-0.15 μmol of sodium formate (determined by the measurement of 2H) was converted to benzyl formate and analyzed by gas chromatography-mass spectrometry as described previously(12.Akhtar, M., Corina, D. L., Miller, S. L., Shyadehi, A. Z., Wright, J. N. (1994) J. Chem. Soc. Perkin Trans. I, 263–267Google Scholar).Incubations under 18O2 GasA procedure similar to that given immediately above was employed except that 18O2 gas was used instead of air in the following manner. The vessel containing all the components but the substrate was evacuated, using a water pump, and flushed with argon. After two rounds of the preceding operation a solution of the substrate was added and the vessel immediately evacuated, flushed with 18O2 (isotopic purity: 97%)/argon (1:2 ratio by volume) and after closing the tap the incubation was performed as above.RESULTSSynthesis of 32-Labeled Precursors for Sterol 14α-Demethylase and Preliminary Enzymic Studies3β-Acetoxylanost-7-en-32-onitrile (5), obtained by a lengthy 10-step sequence as described by Barton and co-workers(13.Barton, D. H. R., McGhie, J. F., Batten, P. L. (1970) J. Chem. Soc. Sect. C Org. Chem. 1033–1042Google Scholar, 14.Batten, P. L., Bentley, T. J., Boar, R. B., Draper, R. W., McGhie, J. F., Barton, D. H. R. (1972) J. Chem. Soc. Perkin Trans. I 739–748Google Scholar), was the crucial intermediate used for the preparation of four isotopomers each of the 32-oxo (6) and 32-hydroxy (7) substrates. The main methodological improvement made in the synthetic protocol (Fig. SII) was the use of diisobutylaluminum hydride, instead of LiAlH4 in the original work for the conversion of the nitrile (5) into aldehyde (6), which decreased the reaction time from 72 to less than 0.5 h.Scheme II:Structure of the key synthetic intermediate (5.Corbin C.J. Graham-Lorence S. McPhaul M. Mason J.I. Mendelson C.R. Simpson E.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8948-8952Crossref PubMed Scopus (295) Google Scholar) and various isotopomers of the 32-oxo (6.Tan L. Muto N. Eur. J. Biochem. 1986; 156: 243-250Crossref PubMed Scopus (70) Google Scholar) and 32-hydroxy derivatives(7.Kalb V.F. Woods C.W. Turi T.G. Dey C.R. Sutter T.R. Loper J.C. DNA (N. Y.). 1987; 6: 529-537Crossref PubMed Scopus (160) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The two tritiated substrates (6e) and (7d) were used for the assay of the 14α-demethylase activity by monitoring the release, in the medium, of 2HCOOH from the aldehyde (6e) or 2HCOOH plus 2H2O from the hydroxy compound (7d). In the metabolism of the hydroxy compound (7d), tritium is released in water during the oxidation of the hydroxy into the aldehyde group and in formic acid during the subsequent C-C bond cleavage step converting the 32-oxo derivative (6) to the 7,14-diene (see Fig. SIII, structure of the type 11). An oxidative activity in most preparations of 14α-demethylase converts the initially produced formate into CO2 and H2O. Our projected mechanistic experiments required an improved enzyme activity, free from the above oxidation reaction, in order to provide at least 4 μg of formic acid for MS analysis.Scheme III:Postulated mechanism for the acyl-carbon bond cleavage reaction catalyzed by sterol 14α-demethylase using 6a as the substrate. The reactions in the sequence are: (i) adduct formation using the FeIII-OOH species, which is formed from the resting state of the enzyme, 2e, O2, and H+; (ii) homolytic cleavage; (iii) fragmentation; and (iv) disprop ortionation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Heterologous Expression of Sterol 14α-Demethylase of C. albicansThe requirement for an improved source of enzyme for the projected study and the importance of sterol 14α-demethylase as a target for the development of antifungal agents prompted experiments on the expression of the enzyme. In our previous studies the vector pW91P containing phosphoglycerate kinase promoter was used for the expression of C. albicans CYP51 gene in S. cerevisiae, and about 100 pmol of enzyme/mg of microsomal protein were obtained(18.Kelly S.L. Arnoldi A. Kelly D.E. Biochem. Soc. Trans. 1993; 21: 1034-1038Crossref PubMed Scopus (80) Google Scholar). Further improvement has now been achieved using GAL10 promoter (19.Guenguerich F.P. Brian W.R. Sari M.-A. Ross J.T. Methods Enzymol. 1991; 206: 130-145Crossref PubMed Scopus (63) Google Scholar) of the vector YEp51 in conjunction with the yeast strain GRF18(20.Scheller U. Kraft R. Schröder K.-L. Schunck W.-H. J. Biol. Chem. 1994; 269: 12779-12783Abstract Full Text PDF PubMed Google Scholar). Under the conditions of growth used in the present study, GRF18 harboring the expression vector without the insert gave undetectable levels of P-450; however, the cells still synthesized ergosterol, indicating a low level of endogenous expression. Although the phosphoglycerate kinase expression system had indicated that functional C. albicans CYP51 is produced from the native gene(18.Kelly S.L. Arnoldi A. Kelly D.E. Biochem. Soc. Trans. 1993; 21: 1034-1038Crossref PubMed Scopus (80) Google Scholar), we rectified the mutation that will occur on expression in S. cerevisiae due to the deviation in the genetic code discovered in C. albicans(21.Santos A.S. Keith G. Tuite M.F. EMBO J. 1993; 12: 607-616Crossref PubMed Scopus (118) Google Scholar). In the latter organism, CTG, the triplet for Leu, is used for the incorporation of Ser. The alteration of the CTG triplet at position 263 to TCT by recombinant PCR was undertaken to allow a Ser to be inserted in this position, as occurs in C. albicans, when the protein is expressed in S. cerevisiae instead of Leu. The cloning strategy is illustrated in Fig. 1 and other details are described under "Experimental Procedures."Transformation of the yeast strain GRF18 with YEp51:CYP51 produced 1.5 μmol of the demethylase/liter of culture, while the derived microsomal fraction was found to contain up to 2.5 nmol of P-450/mg of protein. The level of expression is higher than has been reported for other P-450 in yeast or E. coli, suggesting that the availability of heme is not limiting. This productivity was not dependent on the CTG to TCT mutation undertaken. Molecular modelling studies predict that the residue at position 263 is on the surface of the protein, thus explaining the absence of effect on the activity of the enzyme when the unmodified gene was expressed previously. ( (2)A. Z. Shyadehi, D. C. Lamb, S. L. Kelly, D. E. Kelly, W.-H. Schunck, J. N. Wright, D. Corina, and M. Akhtar, manuscript in preparation.) Table I shows that the specific activity of the enzyme in microsomes from recombinant vector, based on release of formic acid from the 2H-labeled 32-oxo derivative (6e), was 0.1- 0.25 nmol/nmol of P-450/min, and, as expected, no activity was detectable in the host strain harboring the parent vector. The specific activity of the cloned enzyme remained unchanged when it was purified to homogeneity and reconstituted with NADPH-cytochrome P-450 reductase from pig liver. The activity is similar to that found for homogeneous CYP51 obtained from a wild type strain
Referência(s)