©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mechanism of the Acyl-Carbon Bond Cleavage Reaction Catalyzed by Recombinant Sterol 14-Demethylase of Candida albicans (Other Names Are: Lanosterol 14-Demethylase, P-450, and CYP51) (*)

(Received for publication, December 4, 1995; and in revised form, February 21, 1996)

Akbar Z. Shyadehi (1) David C. Lamb (2) Steven L. Kelly (2)(§) Diane E. Kelly (2) Wolf-Hagen Schunck (3) J. Neville Wright (1) David Corina (1) Muhammad Akhtar (1)(§)

From the  (1)Department of Biochemistry, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom, the (2)Department of Molecular Biology and Biochemistry, University of Sheffield, Sheffield S10 2UH, United Kingdom, and the (3)Max Delbrück Center for Molecular Medicine, Robert-Rossle-Strasse 10, D-13122 Berlin-Buch, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
FOOTNOTES
REFERENCES

ABSTRACT

The Candida albicans sterol 14alpha-demethylase gene (P-450, 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 14alpha-demethylase, 3beta-hydroxylanost-7-en-32-al and 3beta-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-O]- and [32-^2H,32-^18O]3beta-hydroxylanost-7-en-32-al with the recombinant 14alpha-demethylase was performed under O(2) or ^18O(2) 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-450) and 17alpha-hydroxylase-17,20-lyase (P-450,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14alpha-demethylase is considered.


INTRODUCTION

Our earlier studies on the removal of C-19 of androgens in the formation of estrogens (1, 2, 3) and of the 14alpha-methyl group of lanosterol during sterol biosynthesis (Fig. SI, Conversion 14) (4) raised the possibility that these seemingly unrelated conversions may occur by closely related mechanisms involving three steps as shown in Fig. R1.



Scheme I: The sequence of reactions catalyzed by sterol 14alpha-demethylase. Although lanosterol is the physiological substrate for the enzyme from most sources, dihydrolanosterol (reduced at the 24,25-double bond) as well as lanostane derivatives containing a Delta7-double bond also serve as substrates. With Delta7-substrates, 7,14-diene is formed following the C-C bond cleavage step.





Figure R1: Reaction 1.




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-450) (5, 6) and lanosterol 14alpha-demethylase (P-450)(7, 8) . The third step in estrogen biosynthesis has aroused much interest (9, 10) and the current view of the mechanism is influenced by our ^18O labeling experiments(2, 3) , 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) .


Figure R2: Reaction 2.




Although all the experimental findings available to date on the C-C bond cleavage step in 14alpha-demethylation, for example the requirement for NADPH plus O(2) for the reaction and release of the C(1) unit as formate, could be explained (3, 9) 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 14alpha-demethylase (34).


EXPERIMENTAL PROCEDURES

Materials

Isotopically enriched ^18O(2) (97%) admixed with 2 volumes of argon was obtained from Isogas Limited, Croydon, Surrey and H(2)^18O 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) . 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 3beta-acetoxylanost-7-en-32-onitrile (5) gave expected melting points, R(F) values, IR spectra, as well as mass spectrometric data.

Gas Chromatography-Mass Spectrometry

Isotopic 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 C 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 times 0.32 mm inner diameter column of DB17 with splitless injection(12) .

The experimentally determined value for C 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.

3beta-Hydroxylanost-7-en-32-al (6a)

A commercially available mixture of lanost-8-en-3beta-ol and lanost-8,24-dien-3beta-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) , lanost-8-en-3beta-ol acetate was converted to 3beta-hydroxylanost-7-one from which was prepared 3beta-acetoxylanost-7-en-32-onitrile (5) by the procedure described by Batten et al.(14) . The nitrile (5) was reduced to 3beta-hydroxylanost-7-en-32-al (6a) either with lithium aluminum hydride (14) or with diisobutylaluminum hydride as outlined below.

3beta-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 times 45 cm), and eluted with petroleum ether containing increasing amounts of ethyl acetate (up to 10%). The solvent was removed in vacuo. The resulting 3beta-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) , R(F), 0.5 (ethyl acetate/petroleum ether, 3:7); IR (Nujol) 3540-3150 and 1720 cm. Mass spectrum of its trimethylsilyl derivative gave a molecular ion at m/z 514.

[32-^2H]3beta-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 3beta-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 (D(1); 88%) and 514 (D(0); 12%).

[32-^2H,32-^18O]3beta-Hydroxylanost-7-en-32-al (6c)

[32-^2H]3beta-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]3beta-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 (D(1), ^18O; 76%), 516 (^18O; 6%), 515 (D(1); 8%), and 514 (D(0); 9%).

[32-^3H]3beta-Hydroxylanost-7-en-32-al(6e)

3beta-Hydroxylanost-7-en-32-al (6a) was converted to the corresponding 3beta-acetoxy-compound (6a; R, CH(3)CO) 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-^3H]3beta-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-^3H]3beta-hydroxylanost-7-en-32-al (23 mg, 0.052 mmol; 15.5 µCi/µmol).

3beta-Hydroxylanost-7-en-32-ol (7a)

To 3beta-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 times 38 cm) with petroleum ether containing increasing amounts of ethyl acetate (up to 20%) to give after crystallization from diethyl ether-petroleum ether 3beta-hydroxylanost-7-en-32-ol (70 mg, 0.16 mmol), m.p. 200-203 °C, R(F) 0.62 (ethyl acetate-petroleum ether, 1:1) and its mass spectrum gave a molecular ion at m/z 444.

[32-^2H(2)]3beta-Hydroxylanost-7-en-32-ol (7b)

This compound was prepared from [32-^2H]3beta-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 (D(2); 78%), 445 (D(1); 13%), and 444 (D(0); 9%).

[32-^2H(2);32-^18O]3beta-Hydroxylanost-7-en-32-ol(7c)

The title compound was obtained from [32-^2H,32-^18O]3beta-hydroxylanost-7-en-3-al (6c) by reduction with sodium borodeuteride, as described above. The recrystallized product had m/z (italics), [composition; % distribution] 448[D(2),^18O; 38%], 447[D(1),^18O; 13%], 448[D(2); 36%], 445[D(1); 1%] and 444[D(0); 12%].

[32-^3H]3beta-Hydroxylanost-7-en-32-ol (7d)

A solution of 3beta-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 Liver

Pig 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 times g for 30 min. The supernatant was subsequently spun at 10,5000 times 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 protein.

Recombinant DNA Manipulations

Our 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) . Higher level expression was achieved by recombinant PCR (^1)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) 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 1: Strategy 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.



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 Transformations

Escherichia coli strain DH5alpha was used for bacterial transformation and plasmid preparation. Yeast transformation to leucine prototrophy used the strain GRF18 (Matalpha leu2-3,2-112 his3-11, 3-15).

Growth of Yeast for Heterologous Expression

Yeast 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 10^8 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 times g for 5 min using a bench centrifuge. The resulting supernatant was centrifuged twice at 10,000 times g for 20 min to remove mitochondria and then at 100,000 times 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) and protein using a Sigma BCA kit.

Determination of Sterol 14alpha-Demethylase Activity of the Microsomes

A 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 times 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 Formate

A 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 3beta-hydroxylanost-7-en-32-al (6) or 3beta-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 times 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 ^3H) was converted to benzyl formate and analyzed by gas chromatography-mass spectrometry as described previously(12) .

Incubations under ^18O(2) Gas

A procedure similar to that given immediately above was employed except that ^18O(2) 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 ^18O(2) (isotopic purity: 97%)/argon (1:2 ratio by volume) and after closing the tap the incubation was performed as above.


RESULTS

Synthesis of 32-Labeled Precursors for Sterol 14alpha-Demethylase and Preliminary Enzymic Studies

3beta-Acetoxylanost-7-en-32-onitrile (5), obtained by a lengthy 10-step sequence as described by Barton and co-workers(13, 14) , 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 LiAlH(4) 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) and various isotopomers of the 32-oxo (6) and 32-hydroxy derivatives(7) .



The two tritiated substrates (6e) and (7d) were used for the assay of the 14alpha-demethylase activity by monitoring the release, in the medium, of ^3HCOOH from the aldehyde (6e) or ^3HCOOH plus ^3H(2)O 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 14alpha-demethylase converts the initially produced formate into CO(2) and H(2)O. 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 14alpha-demethylase using 6a as the substrate. The reactions in the sequence are: (i) adduct formation using the Fe-OOH species, which is formed from the resting state of the enzyme, 2e, O(2), and H; (ii) homolytic cleavage; (iii) fragmentation; and (iv) disprop ortionation.



Heterologous Expression of Sterol 14alpha-Demethylase of C. albicans

The requirement for an improved source of enzyme for the projected study and the importance of sterol 14alpha-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) . Further improvement has now been achieved using GAL10 promoter (19) of the vector YEp51 in conjunction with the yeast strain GRF18(20) . 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) , 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) . 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. 1and 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)

Table 1 shows that the specific activity of the enzyme in microsomes from recombinant vector, based on release of formic acid from the ^3H-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 of C. albicans(22) , but lower than those reported from other sources(8, 23, 24, 25) . The reason for the low specific activity of C. albicans CYP51 is not known but the possibility has been considered that the physiological substrate for this enzyme may be 24-methylene dihydrolanosterol rather than the lanosterol derivatives used in in vitro assays by us and others(22) .



Mass Spectrometric Analysis of Formate Released from the 32-Oxo Derivatives (6) Variously Labeled at C-32 with ^2H and ^18O

Befor e dealing with the results of the enzymic incubations, attention is drawn to the fact that a quantitative determination of the ^18O content of formate produced biosynthetically is fraught with a number of problems. The one that should be mentioned from the very outset is the dilution of the biosynthetic formate with the unlabeled species ubiquitously present in reagents, solvents, and enzymic preparations. We have estimated that 5-10 µg of formate is present per ml of a typical incubation, and to circumvent this interference, the precursors used in this study were labeled with deuterium at the position of interest, C-32, so that the ^18O content of only deuterium containing isotopomers of formic acid analyzed as benzyl formate (HCOO-Bzl) was used in the quantitation. Even this approach is not without its problems, since the peak at m/z 137 due to ^2HCOO-Bzl also contains substantial contributions from the C isotopic species present in the unlabeled material. The intensity of the m/z 137 peak and also of peaks with higher masses could be corrected for C natural abundance, but with limited accuracy. In the experiments reported in this paper, the biosynthetically produced formate was between 5 and 7% of the total formate present in the sample, and the corrected intensities of peaks are subject to a standard deviation of ±10.

The mass spectrometric analysis, using either full scan or selected ion recording, of benzyl formate obtained from the incubation of the ^2H-labeled aldehyde (6b) under O(2) gave a single isotopic peak at m/z 137 due to ^2HCOO-Bzl (entry 1, Table 2). The absence of molecular ions due to higher masses m/z 138-139 established that t he important region of the spectrum was free from background noise. Benzyl formate from the incubation of the ^2H-labeled aldehyde (6b) under ^18O(2) gave a major peak at m/z 139 ascribable to ^2HC^18OO-Bzl (entry 2, Table 2). The intensity of the la tter peak was at least 50% of the combined intensities of the peaks due to all the ^2H-containing isotopomers of benzyl formate. This result shows that during the cleavage of the C-14-C-32 bond of the aldehyde (6) one atom of oxygen from ^18O(2) is incorporated into the released format e. Now the complementary experiment was performed in which the doubly labeled aldehyde (6c), containing ^2H as well as ^18O at C-32, was used as the substrate and incubated under O(2). Under these conditions, a predominant peak at m/z 139 was observed (entry 3, Table 2), showing the transfer of the carbonyl oxygen of the aldehyde into the formate. The most significant feature of the experiment in which the same doubly labeled precursor (6c) was deformylated under ^18O(2) was the presence of a peak at m/z 141 for the isotopomer in which the deuterium containing benzyl formate contained two atoms of ^18O. In essence, this experiment (entry 4, Table 2) represents the summation of the results of entries 2 and 3, showing that in the cleavage of the C-C bond of the aldehyde by the 14alpha-demethylase the original aldehydic oxygen of the substrate is retained in the released formic acid, while its second oxygen is derived from molecular oxygen.



Attention is drawn to the presence of substantial amounts of deuteriated benzyl formate containing either one or no^18O in the experiment of entry 4 (Table 2). The formation of these species is attributed to the loss of the aldehydic oxygen by exchange with the oxygen of H(2)O during the incubation. The extent of the exchange increases in the experiments performed under ^18O(2) because of the need to perform time-consuming manipulations for replacing air with ^18O gas. Another adverse consequence of this operation is some denaturation of the enzyme resulting in the production of lower amounts of formate.


DISCUSSION AND CONCLUSIONS

The notion that the C-C bond cleavage reaction in the multistep process catalyzed by 14alpha-demethylase occurs by the same generic reaction that has previously been found to operate for aromatase (CYP19) (2, 3) and 17alpha-hydroxylase-17,20-lyase (CYP17) (12) is supported by the present study. The fission process corresponding to an acyl-carbon cleavage is reducible to the stoichiometry of Fig. R2. The two main predictions of Fig. R2, that in the overall process the carbonyl oxygen atom of the substrate, together with an atom of oxygen from O(2), are incorporated in the expelled formate, have been validated experimentally. We have advocated that the cleavage process may be rationalized by assuming that in the catalytic cycle of P-450s, the Fe-OOH species, which is normally directed to produce an iron-monooxygen species involved in the hydroxylation reaction, may be trapped to give an adduct when the substrate skeleton contains a properly juxtapositioned electrophilic center(26, 27) .

The intermediacy of a peroxide adduct in acyl-carbon bond cleavages (Fig. R2) has been inferred from a range of observations, either described or reviewed in previous publications(10, 27, 28, 29, 30) . Several mechanistic alternatives are possible through which the products of the reaction of Fig. R2may be formed from the peroxide-adduct of the type 8, Fig. SIII. For example, in the case of CYP17 evidence has been presented to show that certain acyl-carbon bond cleavage reactions catalyzed by the enzyme occur by a homolytic fission route producing a carbon radical that either undergoes a disproportionation process producing an olefin or an oxygen rebound reaction forming a hydroxy compound(29) . A similar scenario may be envisaged for the related acyl-carbon bond cleavage reaction, promoted by 14alpha-demethylase, as shown in Fig. SIII. When the intermediacy of a peroxide-adduct was originally proposed, the possibility was considered that it may rearrange by a Baeyer-Villiger process to produce a formate ester which then, through an elimination reaction, creates the double bond in the final product(2) . Such a possibility was, however, excluded for aromatase by showing that 10beta-hydroxyestr-4-ene-3,17-dione formate was not aromatizd by the enzyme(2, 9) . O-Acyl derivatives have been isolated during the reactions catalyzed by 14alpha-demethylase (31) and CYP17(32) , but further work is required to establish whether these are bona fide intermediates in the acyl-carbon cleavage reaction or are formed merely as side products. The problem posed by a mechanism for the reaction of Fig. R2, which operates through the intermediacy of an O-acyl derivative is that it requires a single enzyme, not only to possess the activity for three different oxidative reactions, but also a fourth activity to promote the difficult removal of the elements of formic acid.

In the light of these considerations, we favor the mechanism shown in Fig. SIIIfor the conversion of the 32-oxo derivative (6) into the 7,14-diene (11). In principle the initially formed peroxy adduct (8) may cleave by an ionic or a radical process. The latter cleavage mode, however, has the advantage that it gives an intermediate alkoxy radical (9), which is ideally suited to undergo fragmentation producing formate, and the substrate radical (10), which can be conveniently converted into the product (11). Furthermore, the mechanism is based on a precedent from an equivalent acyl-carbon cleavage reaction catalyzed by CYP17 for which evidence for a radical process has been obtained (29) .


FOOTNOTES

*
This work was supported by a Science and Engineering Research Council (now Engineering & Physical Sciences Research Council and Biotechnology & Biological Sciences Research Council) research grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Corresponding authors for the mechanistic and cloning work are M. Akhtar and S. L. Kelly, respectively: Dept. of Biochemistry, University of Southampton, Bassett Crescent East, Southampton, Hants. SO16 7PX, UK. Tel.: 44-01703-594323; Fax: 44-01703-594459.

(^1)
The abbreviation used is: PCR, polymerase chain reaction.

(^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.


REFERENCES

  1. Skinner, S. J. M., and Akhtar, M. (1969) Biochem. J. 114, 75-81 [Medline] [Order article via Infotrieve]
  2. Akhtar, M., Calder, M. R., Corina, D. L., and Wright, J. N. (1982) Biochem. J. 201, 569-580 [Medline] [Order article via Infotrieve]
  3. Stevenson, D. E., Wright, J. N., and Akhtar, M. (1988) J. Chem. Soc. Perkin Trans. I 2043-2052
  4. Akhtar, M., Alexander, K., Boar, R. B., McGhie, J. F., and Barton, D. H. R. (1978) Biochem. J. 169, 449-463 [Medline] [Order article via Infotrieve]
  5. Corbin, C. J., Graham-Lorence, S., McPhaul, M., Mason, J. I., Mendelson, C. R., and Simpson, E. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8948-8952 [Abstract]
  6. Tan, L., and Muto, N. (1986) Eur. J. Biochem. 156, 243-250 [Abstract]
  7. Kalb, V. F., Woods, C. W., Turi, T. G., Dey, C. R., Sutter, T. R., and Loper J. C. (1987) DNA (N. Y.) 6, 529-537 [Medline] [Order article via Infotrieve]
  8. Aoyama, Y., Yoshida, Y., Sonoda, Y., and Sato, Y. (1987) J. Biol. Chem. 262, 1239-1243, and references therein [Abstract/Free Full Text]
  9. Akhtar, M., Njar, V. C., and Wright, J. N. (1993) J. Steroid Biochem. Mol. Biol. 44, 375-387 [CrossRef][Medline] [Order article via Infotrieve]
  10. Oh, S. S., and Robinson, C. H. (1993) J. Steroid Biochem. Mol. Biol. 44, 389-397 [CrossRef][Medline] [Order article via Infotrieve]
  11. Corina, D. L., and Dunstan, P. M. (1973) Anal. Biochem. 53, 571-578 [Medline] [Order article via Infotrieve]
  12. Akhtar, M., Corina, D. L., Miller, S. L., Shyadehi, A. Z., and Wright, J. N. (1994) J. Chem. Soc. Perkin Trans. I , 263-267
  13. Barton, D. H. R., McGhie, J. F., and Batten, P. L. (1970) J. Chem. Soc. Sect. C Org. Chem. 1033-1042
  14. Batten, P. L., Bentley, T. J., Boar, R. B., Draper, R. W., McGhie, J. F., and Barton, D. H. R. (1972) J. Chem. Soc. Perkin Trans. I 739-748
  15. Kelly, S. L., Lamb, D. C., Corran, A. J., Baldwin, B. C., and Kelly, D. E. (1995) Biochem. Biophys. Res. Commun. 207, 910-915 [CrossRef][Medline] [Order article via Infotrieve]
  16. Lai, M. H., and Kirsch, D. R. (1989) Nucleic Acids Res. 17, 804 [Medline] [Order article via Infotrieve]
  17. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2370-2378 [Free Full Text]
  18. Kelly, S. L., Arnoldi, A., and Kelly, D. E. (1993) Biochem. Soc. Trans. 21, 1034-1038 [Medline] [Order article via Infotrieve]
  19. Guenguerich, F. P., Brian, W. R., Sari M.-A., and Ross, J. T. (1991) Methods Enzymol. 206, 130-145 [Medline] [Order article via Infotrieve]
  20. Scheller, U., Kraft, R., Schröder, K.-L., and Schunck, W.-H. (1994) J. Biol. Chem. 269, 12779-12783 [Abstract/Free Full Text]
  21. Santos, A. S., Keith, G., and Tuite, M. F. (1993) EMBO J. 12, 607-616 [Abstract]
  22. Hitchcock, C. A., Dickenson, K., Brown, S. B., Evans, E. G. V., and Adams, D. J. (1989) Biochem. J. 263, 573-579 [Medline] [Order article via Infotrieve]
  23. Trzaskos, J., Kawata, S., and Gaylor, J. L. (1986) J. Biol. Chem. 261, 14651-14657 [Abstract/Free Full Text]
  24. Sano, H., Sonoda, Y., and Sato, Y. (1991) Biochim. Biophys. Acta 1078, 388-394 [Medline] [Order article via Infotrieve]
  25. Sonoda, Y., Endo, M., Ishida, K., Sato, Y., Fukusen, N., and Fukuhara, M. (1993) Biochim. Biophys. Acta 1170, 92-97 [Medline] [Order article via Infotrieve]
  26. Akhtar, M., and Wright, J. N. (1991) Nat. Prod. Rep. 527-551
  27. Akhtar, M., Wright, J. N., Shyadehi, A. Z., and Robichaud, P. (1994) Pure Appl. Chem. 66, 2387-2390
  28. Akhtar, M., Corina D., Miller, S., Shyadehi, A. Z., and Wright, J. N. (1994) Biochemistry 33, 4410-4418 [Medline] [Order article via Infotrieve]
  29. Lee-Robichaud, P., Shyadehi, A. Z., Wright, J. N., Akhtar, M. E., and Akhtar, M. (1995) Biochemistry 34, 14104-14113, and references therein [Medline] [Order article via Infotrieve]
  30. Vaz, A. D. N., Roberts, E. S. & Coon, M. J. (1991) J. Am. Chem. Soc. 113, 5886-5887
  31. Fisher, R. T., Trzaskos, J. M., Magolda, R. L., Ko, S. S., Brosz, C. S., and Larsen, B. (1991) J. Biol. Chem. 266, 6124-6132 [Abstract/Free Full Text]
  32. Swinney, D. C., and Mak, A. Y. (1994) Biochemistry 33, 2185-2190 [Medline] [Order article via Infotrieve]

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