Oxygenation cascade in conversion of n-alkanes to alpha,omega-dioic acids catalyzed by cytochrome P450 52A3.

Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system. Besides the primary product, 1-hexadecanol, the conversion of hexadecane yielded up to five additional metabolites, which were identified by gas chromatography-electron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1, 16-hexadecanediol, 16-hydroxyhexadecanoic acid, and 1, 16-hexadecanedioic acid. As shown by substrate binding studies, the final product 1,16-hexadecanedioic acid acts as a competitive inhibitor of n-alkane binding and may be important for the metabolic regulation of the P450 activity. Kinetic studies of the individual sequential reactions revealed high Vmax values for the conversion of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min-1, respectively), whereas the oxidation of hexadecanoic acid, 1, 16-hexadecanediol, and 16-hydroxyhexadecanoic acid occurred at significantly lower rates (9, 9, and 5 min-1, respectively). 1-Hexadecanol was found to be the main branch point between mono- and diterminal oxidation. Taken together with data on the incorporation of 18O2-derived oxygen into the hexadecane oxidation products, the present study demonstrates that a single P450 form is able to efficiently catalyze a cascade of sequential mono- and diterminal monooxygenation reactions from n-alkanes to alpha, omega-dioic acids with high regioselectivity.

The capability of several yeast species to use n-alkanes and other aliphatic hydrocarbons as a sole source of carbon and energy is mediated by the existence of multiple microsomal cytochrome P450 forms. Corresponding P450 genes or cDNAs have been isolated from the alkane-assimilating yeasts Candida maltosa (1)(2)(3)(4)(5)(6), C. tropicalis (7)(8)(9), and C. apicola (10). The presently available 21 sequences belong to the family CYP52, according to the nomenclature of the P450 superfamily (11).
The metabolic functions of P450 in these yeasts have been established thus far in the terminal oxidation of long-chain n-alkanes to fatty alcohols as the first and rate-determining step of the n-alkane degradation pathway and in the -hydroxylation of fatty acids, initiating the diterminal degradation pathway (Refs. 12-14; for a review, see Ref. 15). Sequential gene disruption revealed that in C. maltosa, four of its eight CYP52 genes, namely CYP52A3, CYP52A4, CYP52A5, and CYP52A9, are directly involved in alkane assimilation (16). After heterologous expression in Saccharomyces cerevisiae, each of the corresponding P450 isoenzymes was found to exhibit an individual substrate specificity in terms of the preferred class (n-alkanes or fatty acids) and the chain length of the hydroxylated aliphatic compounds (17,18).
In the present study, we addressed the question of whether P450 monooxygenases may be involved not only in the primary hydroxylation reactions mentioned above but also in the subsequent oxidation steps leading to the formation of fatty acids and long-chain dioic acids, which are then subjected to peroxisomal ␤-oxidation. For that purpose, P450 52A3, 1 which represents the major alkane-inducible P450 form in C. maltosa (16,19), was selected. Its substrate specificity is characterized by a preferential binding and hydroxylation of n-alkanes compared with fatty acids and by a general chain length optimum for such compounds with 16 carbon atoms (3,17). Therefore, hexadecane and the putative oxidation intermediates on the way to 1,16-hexadecanedioic acid were used as model substrates, and the kinetics of their P450 52A3-catalyzed conversion and the incorporation of 18 O 2 -derived oxygen into the products were studied. To avoid potential contaminations with other P450 forms or additional C. maltosa enzymes, P450 52A3 and the corresponding NADPH-P450 reductase were heterologously expressed in S. cerevisiae, purified to electrophoretic homogeneity, and reconstituted into an active enzyme system. The results clearly demonstrate that P450 52A3 is able to catalyze the complete oxidation of n-alkanes to ␣,-dioic acids, and that this conversion is based on sequential mono-and diterminal monooxygenation reactions.

EXPERIMENTAL PROCEDURES
Materials-[1-14 C]Hexadecane, 1-[1-14 C]hexadecanol, and [1-14 C]hexadecanoic acid with specific activities of 57, 54, and 56.6 mCi/mmol, respectively, were purchased from either Sigma (Deisenhofen, Germany) or Amersham-Buchler (Braunschweig, Germany). [1-14 C]Hexadecanal was produced by enzymatic oxidation of labeled 1-hexadecanol using the peroxisomal fatty alcohol oxidase from the yeast C. maltosa as described previously (14). 1,16-[ 14 C]Hexadecanediol and 16-[ 14 C]hydroxyhexadecanoic acid were prepared by the P450 52A3-catalyzed hydroxylation of labeled 1-hexadecanol in a reconstituted system composed as described below. 18 O 2 gas (95 atom %) was purchased from Linde (Mü nchen, Germany), and tert-butyldimethyl-chlorosilane and N-nitroso-N-methyl-p-toluenesulfonamide were obtained from Merck (Darmstadt, Germany). P450 52A3 and NADPHcytochrome P450 reductase (CPR) from C. maltosa were heterologously expressed in S. cerevisiae GRF18 and purified to electrophoretic homogeneity as described previously (17,20,21).  18 O 2 were carried out as described above, except that: (i) the incubation system consisted of a vial sealed with a rubber stopper and plastic seal, and (ii) it was evacuated several times, flushed with nitrogen, and filled with 18 O 2 from a low pressure steel flask by means of a three-way valve. After evacuation and the introduction of 18 O 2 , the reaction was started by the injection of NADPH in a solution that was previously evacuated and flushed with nitrogen.

Reconstitution and Enzyme Assay for Product Characterization-For
Reconstitution and Enzyme Assay for Kinetic Studies-For kinetic studies on the hydroxylation of hexadecane and the individual intermediates of the hexadecane degradation pathway, the enzyme system was reconstituted from the purified protein components by the cholate dialysis method in the presence of the total microsomal phospholipid fraction of S. cerevisiae, as described previously (17).
Substrate Binding Studies-Spectrophotometric titration of purified P450 52A3 with C 16 substrates was performed at different substrate concentrations ranging from 0.03 to 0.50 mM, as described previously (17). For inhibitory studies on hexadecane binding, substrate difference spectra were also recorded in the presence of 0.125, 0.25, and 0.5 mM 1,16-hexadecanedioic acid.
Product Analysis-Experimental conditions for product extraction, separation by TLC, and determination of the enzyme activity were as described previously (17). For preliminary product identification, the R F values of the radiolabeled hexadecane metabolites were compared with those of authentic standards visualized with 2Ј,7Ј-dichlorofluorescein under UV light.
For final product identification by GC-EIMS as well as the preparation of stock solutions of commercially unavailable 14 C-labeled substrates, each marked spot was cut off from the TLC plate, and the corresponding product was then extracted with chloroform/methanol (1:2); the extract was evaporated, and the residue was redissolved in methanol. Before GC-EIMS analysis, the following derivatization reactions were performed: (i) methylation of hexadecanoic acid and 16hydroxyhexadecanoic acid with diazomethane (22), and (ii) silylation of both the methyl ester of 16-hydroxyhexadecanoic acid and 1-hexadecanol with tert-butyl-dimethyl-chlorosilane/imidazole reagents (23). GC-EIMS was recorded at 70 eV with a Fisons GC 8000 quadrupole gas chromatograph coupled with a Fisons MD 800 mass spectrometer. The experiments were carried out with injector, ion source, and interface temperatures of 230°C, 220°C, and 250°C, respectively. Samples in methanol (1 l) were injected in the splitless mode and run on a capillary column (coating thickness, 60 m ϫ 0.25 mm; 95% dimethylsiloxane cross-linked with 5% phenylmethylsiloxane, DB5 MS; J & W Scientific). The oven temperature was programmed for 2 min at 110°C and then increased linearly to 300°C at a rate of 15°C/min. The carrier gas was helium. Fig. 1A, the oxidation of hexadecane by P450 52A3 yielded five metabolites that were detectable as radioactive spots by TLC analysis and were preliminarily identified by a comparison of their R F values with those of unlabeled standards. Hexadecanol and hexadecanoic acid with R F values of 0.44 and 0.57 were found as the main products. Hexadecanediol, hydroxyhexadecanoic acid, and hexadecanedioic acid with R F values of 0.18, 0.25, and 0.34 were detected as additional products. As shown in the time course (Fig.  1B), hexadecane was initially oxidized to hexadecanol, which reached a steady-state level after about 10 min, obviously due to its further oxidation to secondary products.

Oxidation of Hexadecane by P450 52A3-As shown in
Product Identification by GC-EIMS-For identification of the hexadecane oxidation products detected by radio-TLC, the corresponding spots were extracted from the silica plate and subjected to GC-EIMS analysis. The products were identified by a comparison of their retention times and mass fragmentation pattern with those of authentic standards (Fig. 2).
1-Hexadecanol Formation-The major product of hexadecane oxidation, which comigrated in TLC with the hexadecanol standard, was silylated at the hydroxyl group by treatment with the tert-butyl-dimethyl-chlorosilane/imidazole reagent. The derivative showed a single peak at 17.4 min in GC-EIMS with a fragment ion of (M-57) ϩ at m/z 299 due to the scission at the tert-butyl group ( Fig. 2A). Selected ion monitoring carried out at m/z 299 revealed only one peak with a retention time in GC that corresponded exactly to that of the silyl derivative of the 1-hexadecanol standard (data not shown). Products of subterminal hydroxylation were not detectable, confirming the high regioselectivity of P450 52A3. Hexadecanoic Acid Formation-The fragmentation pattern of the second major product of hexadecane oxidation, which comigrated in TLC with the hexadecanoic acid standard, is shown as methylated derivative in Fig. 2B. After methylation, a single peak was detected in GC at 15.2 min that showed the molecular ion at m/z 270. This ion is consistent with the molecular ion of the methyl ester of hexadecanoic acid (24). Typical diagnostic fragments with m/z 74 (McLafferty rearrangement), 87 (␥-cleavage), 143, and 227 were also detected for both the metabolite and the authentic hexadecanoic acid standard.
1,16-Hexadecanediol Formation-The formation of 1,16hexadecanediol was verified without further derivatization by its retention time in GC (17.5 min) and its mass fragment pattern, which was identical to that of the authentic underivatized standard compound. As shown by the mass spectra (Fig. 2D), a diagnostic fragment ion at m/z 31 was detected, indicating the cleavage of -CH 2 OH, which is common to primary alcohols (24). Although the mass spectra of underivatized 1-hexadecanol and 1,16-hexadecanediol were found to be very similar, their retention times in GC differed by more than 3 min, and they could therefore be distinguished.
16-Hydroxyhexadecanoic Acid Formation-After silylation of the methyl ester derivative of a product comigrating in TLC with 16-hydroxyhexadecanoic acid, this compound yielded a single peak at 20.4 min in GC. It showed strong signals of fragment ions corresponding to (M-57-32) ϩ at m/z 311 due to the scissions at the tert-butyl and methanol groups and (M-57) ϩ at m/z 343 due to the scission at the tert-butyl group (Fig.  2C). No additional fragment ions of [CH 3 (CH 2 ) n -CH-O-tert-BDMS] ϩ (n Ն 0) were detectable by selected ion monitoring carried out for hexadecanoic acid at m/z 311 (data not shown). This demonstrated that subterminal hydroxylation did not occur with P450 52A3.
1,16-Hexadecanedioic Acid Formation-The formation of 1,16-hexadecanedioic acid was verified by GC-EIMS after its conversion to dicarboxylic acid dimethyl ester by its retention time in GC (17.2 min) and its mass fragment pattern, which were identical to those of the authentic standard. As shown in Fig. 2E, typical methyl ester peaks were found at m/z 283 (M ϩ -31) and m/z 241 (M ϩ -73). In addition to the McLafferty fragment at m/z 74, a fragment ion at m/z 98 was detected, which is specific for long-chain aliphatic dicarboxylic acid dimethyl esters (25).
Kinetics of the Individual Reaction Steps-As shown above, P450 52A3 is capable of catalyzing sequential mono-and diter-minal oxidation steps on an n-alkane substrate. To determine the kinetic parameters of the individual reactions, all intermediates formed during hexadecane oxidation were further considered as individual substrates. Catalytic activities were determined after short reaction times (5 min) to minimize further oxidation of the products formed during the initial oxidation. The kinetic parameters obtained are shown in Table I. The data indicate that the first step of P450 52A3-catalyzed nalkane oxidation resulting in the formation of 1-hexadecanol occurred with a high V max (27 min Ϫ1 ) and a comparatively low apparent K m (54 M). The oxidation of 1-hexadecanol was catalyzed with similar efficiency (V max ϭ 23 min Ϫ1 , K m ϭ 28 M). Two independent prominent products were formed simultaneously within the first 5 min: (i) hexadecanoic acid, and (ii) 1,16-hexadecanediol (Fig. 1, C and D). In addition, hexadecanal was detected in trace amounts by TLC analysis (Fig. 1C). Its formation as a primary product of the monoterminal oxidation of 1-hexadecanol was further confirmed by GC-EIMS (see below). However, direct estimation of the kinetic parameters for the reaction of 1-hexadecanol to hexadecanal failed due to the rapid further oxidation of hexadecanal to hexadecanoic acid as the corresponding secondary product (V max ϭ 69 min Ϫ1 ; Table  I). The V max values of the further oxidation of hexadecanoic acid and 1,16-hexadecanediol to form the common product 16hydroxyhexadecanoic acid were almost identical but were significantly lower when compared with those of the prior oxidation steps (about 9 min Ϫ1 ). However, the corresponding K m values (94 and 66 M for hexadecanoic acid and 1, 16-hexadecanediol) indicated a kinetically slightly favored oxidation of hexadecanediol to hydroxyhexadecanoic acid. The lowest V max value (4.7 min Ϫ1 ) and the highest apparent K m (126 M) were estimated for the oxidation of 16-hydroxyhexadecanoic acid to    (17), the titration of P450 52A3 with hexadecane induced strong type I spectral changes, indicating a high-affinity binding of the primary substrate to the P450 active site. In this study, 1-hexadecanol and hexadecanal were also found to be effective type I substrates for P450 52A3 with K s values of 58 and 18 M. The addition of 1,16-hexadecanediol, 16-hydroxyhexadecanoic acid, or 1,16hexadecanedioic acid did not induce any spectral changes.
To check the possibility of inhibition of the initial steps in hexadecane oxidation caused by an accumulation of the final product, purified P450 52A3 was titrated with hexadecane in the presence of various concentrations of 1,16-hexadecanedioic acid. The resulting spectral effect was measured by means of substrate difference spectrophotometry. As shown in a doublereciprocal plot of the absorbance change ⌬A 389 -421 nm versus the hexadecane concentration (Fig. 3), the linear graphs obtained crossed the y axis at 1/⌬A max , indicating the 1,16-hexadecanedioic acid as a competitive inhibitor of hexadecane binding. From the slope of the corresponding lines, a K i value of 74 M was estimated.
Mechanism of P450 52A3-catalyzed Oxidation of n-Alkanes to Fatty Acids-As studied in detail for the P450 52A3-catalyzed oxidation of 1-hexadecanol, product formation was completely dependent on the presence of P450, CPR, oxygen, and NADPH (Table II). The omission of any of these components as well as the replacement of NADPH by NAD resulted in no observable product formation. The addition of catalase and superoxide dismutase to the reaction mixture altered neither the catalytic activity nor the corresponding product profile, even after longtime incubation (Table II). This indicates that hydrogen peroxide and superoxide, which could be produced in the reconstituted system, were not involved in nonenzymatic reactions outside the active site of P450 52A3 during the individual oxidation steps.
Experiments performed in 18 O 2 -enriched media were carried out to obtain further insight into the mechanism of the P450 52A3-catalyzed oxidation reactions. Three different substrates were chosen to follow 18 O incorporation/retention during their oxidation by the reconstituted system: (i) hexadecane (experiment 1) to follow isotope enrichment during the first oxidation step, (ii) 1-hexadecanol (experiment 2) to examine 18 (Table III; Fig. 4, A and B).
Taking into account the isotope purity of the 18 O 2 gas used (95 atom %), the results indicate that only a few isotope dilutions occurred under these experimental conditions. Oxidation of 1-Hexadecanol to Hexadecanal-The formation of this aldehyde product was only detected by TLC when using 1-hexadecanol, and not hexadecane, as a substrate. Therefore, unlabeled 1-hexadecanol and 1-[ 18 O]hexadecanol were used to follow 18 O 2 incorporation (experiment 2) and retention (experiment 3; Table III). A comparison of the retention time and the mass fragmentation pattern in GC-EIMS to those obtained for the hexadecanal standard clearly revealed the formation of hexadecanal as a primary product of hexadecanol oxidation. Interpretation of the mass spectra shown in Fig. 4C indicated that two fragment ions could serve to determine the extent of 18 O incorporation into hexadecanal: (i) the mass fragment at m/z 29 corresponding to H-CϵO ϩ , and (ii) the mass peak at m/z 44 due to scission at the ␤-bond close to the aldehyde group accompanied by a shift of one hydrogen to the oxygen-containing fragment (McLafferty rearrangement; Refs. 26 and 27). The mass spectra of hexadecanal formed both in the presence of 18 O 2 from unlabeled 1-hexadecanol (experiment 2) and 1-[ 18 O]hexadecanol (experiment 3) did not show any significant enrichment of 18 O in the carbonyl group. This is indicated by the complete absence of fragment ions at m/z 31 and 46, i.e. two mass units heavier (Fig. 4, C and D). The 18 O label in the residual 1-[ 18 O]hexadecanol remained completely unchanged as detected by mass spectrum analysis after its extraction from the reaction mixture (data not shown).
Oxidation of Hexadecanal to Hexadecanoic Acid-The results obtained from experiments 2 and 3 indicate that enzymatically formed hexadecanal is obviously rapidly hydrated in the incubation medium, and on the time scale of these experiments, the oxygen atom is completely exchanged with solvent water. Hexadecanal is further oxidized to hexadecanoic acid (Table I) (Table III, Fig. 4F).  18 O content of the products was determined from the peaks of the diagnostic fragment ions of the corresponding mass spectra (compare Fig. 4).

Experiment no./substrate Gas
Relative intensities of peaks corresponding to: A reconstituted system consisting of purified recombinant P450 52A3 and CPR was used to define the enzymatic functions of this P450 with respect to the n-alkane metabolism of C. maltosa. The main result is that a single P450 enzyme can efficiently catalyze not only the terminal hydroxylation of longchain n-alkanes and the -hydroxylation of fatty acids as previously suggested (12)(13)(14) but also the complete sequence of mono-and diterminal oxidation steps finally yielding ␣,-dioic acids. This conclusion is based on the product pattern arising from the time course of P450 52A3-catalyzed hexadecane oxidation and the observation that each of the intermediates of the reaction cascade can serve as a separate P450 substrate.
Based on the components required for the oxidation reactions and on quantitative measurements of oxygen incorporation derived from 18 O 2 into the primary and secondary products of hexadecane oxidation, it can be proposed that all steps proceed according to a P450-catalyzed monooxygenase reaction (Fig. 5). As expected, this is clearly the case for the first hy-  droxylation of the terminal methyl groups, leading to the formation of 1-hexadecanol and 1,16-hexadecanediol. The subsequent formation of the aldehyde is also completely dependent on the presence of molecular oxygen, NADPH, and P450 and is assumed to proceed via a hydroxylation of the -CH 2 OH group to a gem-diol. Such a reaction type was clearly demonstrated for the generation of aldehydes from benzylic alcohols by P450s 2B4 and 2E1 (28). Additional examples in which multiple P450catalyzed hydroxylations at one carbon position should lead to gem-diol intermediates are the 14␣-demethylation of lanosterol (29), the C-19 demethylation in estrogen biosynthesis (30), and, more directly related to the present study, the oxidation of (-n)-and of -hydroxy fatty acids to ketones and aldehydes by P450 102 (31) and P450 4A1 (32). In the present study, direct evidence for an incorporation of oxygen from 18 O 2 in the course of aliphatic aldehyde formation could not be obtained. This was obviously due to a rapid exchange with solvent water, which would also explain the loss of 18 O from the labeled hexadecanol upon further oxidation. The mechanism for the subsequent palmitic acid formation is assumed to involve a monooxygenation of the putative gem-diol to an ortho acid intermediate, followed by its dehydration and the release of the carboxylic group. The possible implication of a gem-diol as a substrate for this reaction was already postulated for the oxidation of a xenobiotic aldehyde (11-oxo-⌬ 8 -tetrahydrocannabiol) to a carboxylic acid as catalyzed by P450 2C29 (33). In the present study, the extent of 18 O incorporation into the carboxylic group was significantly lower than the theoretically expected 2:1 ratio between RCO 18 OH and RCOOH. The experimentally determined 1:2 ratio indicates that there is an uneven probability for the loss of either oxygen in the dehydration of the ortho acid, suggesting that this process occurs with some stereoselectivity in the P450 active site, in a similar manner to that proposed for the dehydration of nascent gem-diols (28).
The ability of P450 52A3 to efficiently catalyze an oxygenation cascade on a given n-alkane substrate provides new insights into the metabolic function of P450 monooxygenases in alkane-assimilating yeasts and leads us to propose a partially modified scheme of the alkane degradation pathway (Fig. 6). In accordance with previous models (15, 34 -36), the main route of degradation should include a channeling of fatty alcohols produced by P450s in the endoplasmic reticulum to the fatty alcohol oxidase (37) in the peroxisomes. This process may be facilitated by the close contact of the two membrane compartments revealed by electron microscopy (38). The other oxidation steps now shown to be catalyzed by P450 could be important in providing fatty acids for lipid biosynthesis. Moreover, they allow by-passing of the peroxisomal fatty alcohol oxidase and fatty aldehyde dehydrogenase (39) in producing fatty acids and/or dicarboxylic acids for ␤ oxidation. This, together with the multiplicity of the P450 forms (see the "Introduction"), may lead to a "complementation" of the substrate specificities of the different enzymes involved and could be the basis for the efficient growth of C. maltosa on a broad chain-length range of n-alkanes (C7 to C40).
An additional feature of the new scheme is that it includes several possibilities for a metabolic regulation of P450 activities based on substrate/product competitions. As shown by the results of substrate difference spectra, the dicarboxylic acid clearly acts as a competitive inhibitor of n-alkane binding. Such end product inhibition may be an important element in con-trolling P450 activity and adjusting it to the rate of transport of intermediates out of the endoplasmic reticulum.