INTRODUCTION

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Alkanes as metabolites from organisms (10) and constituents of petroleum (49) and its refined products are widespread compounds in our environment. Containing exclusively apolar -bonds, alkanes are among the chemically least reactive compounds. Bacteria and fungi that utilize alkanes as growth substrates in the presence of molecular oxygen have been known for about 100 years (14, 15). Aerobic microorganisms all initiate the metabolism of alkanes by monooxygenase reactions. These enzymes generate a highly reactive oxygen species by partial reduction of O₂, resulting in the introduction of a hydroxyl group into the alkane molecule by a radical mechanism (22, 51). The alkanol formed is further oxidized and metabolized via the -oxidation pathway.

It was shown only relatively recently that special types of bacteria, which are physiologically and phylogenetically distinct from previously described aerobic hydrocarbon-degrading microorganisms, degrade n-alkanes under strict exclusion of oxygen. n-Alkanes were anaerobically oxidized in pure cultures using sulfate (2, 3, 41, 43) or nitrate (18) as the electron acceptor or in enrichment cultures with sulfate (16) or nitrate (13, 39). Also, anaerobic conversion of long-chain n-alkanes to methane and CO₂ in associations of enriched bacteria and archaea was demonstrated (4, 55). Oxidation of the isoprenoid-like alkane 2,5,9,13-tetramethylpentadecane (pristane) was demonstrated in a nitrate-reducing bacterial community (13). None of the hitherto isolated alkane-degrading anaerobic bacteria utilize aromatic hydrocarbons such as toluene, whereas anaerobic bacteria that degrade aromatic hydrocarbons do not utilize alkanes (24).

First investigations into the anaerobic metabolism of n-alkanes were conducted with two phylogenetically related sulfate-reducing bacteria, strains Hxd3 and Pnd3 (3). Adaptation studies and analysis of cellular fatty acids with strain Hxd3 suggested that anaerobic degradation of alkanes does not occur via desaturation to 1-alkenes, a questionable mechanism that has been speculated about in some early studies on the possibility of anaerobic n-alkane degradation (for an overview, see reference 3). Strain Hxd3 formed mainly C-odd cellular fatty acids during growth with n-hexadecane and C-even cellular fatty acids during growth with n-heptadecane. One explanation for these fatty acid patterns was the assumption of an alteration in the carbon chain length during the initial anaerobic reactions by a C-odd carbon unit, e.g., by terminal addition of a one-carbon moiety. In strain Pnd3, however, cellular fatty acids were mainly C-even upon growth with C-even alkanes and mainly C-odd upon growth with C-odd alkanes; in addition, unidentified fatty acids were formed. The findings suggested different modes of initial reactions in the two n-alkane-degrading sulfate-reducing strains (3). Still, a common principle in the mechanism of initial reactions of n-alkanes in both strains was considered by assuming that the site of carbon addition may also be the subterminal position in the chain (3). In a third isolate of an n-alkane-degrading sulfate-reducing bacterium (strain AK-01), the n-alkanes serving as growth substrates influenced the pattern of cellular fatty acids in a similar manner as in strain Pnd3; in addition, 2-, 4-, and 6-methyl-branched fatty acids were identified (44). By labeling studies, the methyl branch of the fatty acids was shown to be the original terminal carbon of the n-alkane, suggesting addition of a carbon compound to the subterminal position (carbon-2) of the n-alkanes; the carboxyl group was not derived from bicarbonate (44).

So far, no metabolites have been detected that could represent the direct product of the initial anaerobic reaction of an n-alkane. The present study was undertaken to identify such a metabolite and to gain insights into the mechanism of its formation. For the experiments, the recently isolated denitrifying strain HxN1 (18) was chosen. In contrast to other n-alkane-degrading anaerobic bacteria, strain HxN1 grows relatively rapidly (doubling time of 11 h under optimal conditions) and does not adhere to the insoluble alkane phase, so that cells can be harvested easily. Strain HxN1 utilizes n-alkanes with relatively short chains (C₆ through C₈) that are oxidized to CO₂; other anaerobic n-alkane-degrading strains grow preferentially with chain lengths between C₈ and C₁₈. Recently, two-dimensional gel electrophoresis of cell extracts of strain HxN1 revealed specific formation of proteins during growth on n-hexane that were not formed on n-hexanoate. These proteins were therefore supposed to be specifically involved in initial reaction steps of n-alkanes. In the N terminus, one of these proteins exhibited a similarity to the small subunit (BssC) of benzylsuccinate synthase in denitrifying bacteria (A. Behrends, P. Ehrenreich, J. Heider, T. Hurek, S. Ratering, and F. Widdel, unpublished data). This enzyme activates toluene anaerobically by addition of the methyl group to fumarate, yielding benzylsuccinate (24), and there is evidence for the involvement of a glycyl radical in this reaction (17, 31; C. Leutwein, A. J. Pierik and J. Heider, personal communication). Hence, we expected a parallel between the mechanism of the initial reaction of n-alkanes and that of toluene in anaerobic bacteria. Still, a reaction of an alkane in the absence of oxygen would present a novel type of biochemical mechanism, because a saturated hydrocarbon is much less reactive than toluene. In this paper, we report the identification of (1-methylpentyl)succinate (MPS) formed during strictly anaerobic growth of strain HxN1 with nitrate and with n-hexane as the only organic substrate. Furthermore, an electron paramagnetic resonance (EPR) signal characteristic of a glycyl radical was detected in n-hexane-grown cells of strain HxN1. We therefore propose the formation of MPS from n-hexane and fumarate via a radical mechanism as the initial reaction in strain HxN1.

	MATERIALS AND METHODS

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Organism and cultivation. The denitrifying strain HxN1 has been subcultured since its isolation from an enrichment culture (18). It was cultivated under anoxic conditions in chemically defined, bicarbonate-buffered medium with nitrate (9 mM) as the electron acceptor, as described previously (18, 38). Freshly prepared sodium ascorbate (4 mM) was added as the reductant and allowed to scavenge traces of oxygen by incubation for >1 day of the medium before anaerobic inoculation (18). Cultures were grown in stopper-sealed flat glass bottles (500 ml) containing 400 ml of medium under a head space of N₂-CO₂ (90:10, vol/vol). Since pure n-hexane is inhibitory, it was diluted (5% [vol/vol]) in sterile, deaerated 2,2,4,4,6,8,8-heptamethylnonane as an inert, nontoxic carrier phase (18) before addition to the culture (10 ml of mixture per bottle) by means of an N₂-flushed plastic syringe; [1-¹³C]n-hexane was applied in the same manner. d₁₄-n-Hexane was mixed with an equal volume of unlabeled n-hexane, and the isotopomer mixture was applied in the carrier phase like unlabeled n-hexane alone. The hydrocarbons were filter sterilized (regenerated cellulose, 0.2-µm pore size; Schleicher & Schuell, Dassel, Germany) before addition. Sodium n-hexanoate (caproate) in control experiments was added from an autoclaved stock solution (final concentration, 3 mM). Cells were passaged (inoculum size, 5% [vol/vol]) with n-hexane or n-hexanoate as organic substrates at least 10 times before the cultures for the experiments described were inoculated. Cultures with n-hexane were incubated on a slowly rotating shaker (50 rpm) nearly horizontally, with the orifices below the medium surface, so that contact of the overlying, hydrophobic n-hexane/carrier phase mixture with the stoppers was avoided. The incubation temperature was 28°C. In labeling experiments with potential cosubstrates, cultures were grown on n-hexane to an optical density at 660 nm (OD₆₆₀) of 0.1. Then, disodium 2,3-d₂-fumarate or 2,2,3,3-d₄-succinate was added (final concentration, 10 mM), and the culture was allowed to grow to a higher OD. All cultures were used for the described investigations (see following sections) immediately after depletion of nitrate and formed nitrite; the final OD₆₆₀ was usually 0.3 to 0.4. The presence of nitrate and nitrite was examined by using disposable test strips (Merck, Darmstadt, Germany).

Analysis of metabolites. Cultures for metabolite analysis were heated in closed bottles in a water bath to 85°C for 15 min to inactivate eventually present enzymes that may catalyze reactions with oxygen during further handling in the air. Also, release (or additional release) of metabolites into the medium was expected from this treatment. In the case of n-hexane-grown cultures, the aqueous phase was separated from the overlying carrier phase via a separatory funnel. The heated cells were removed by centrifugation (7,000 × g, 20 min) to avoid possible interference with phase separation during subsequent ether extraction. The supernatant from each culture (400 ml) was extracted with diethyl ether (80 ml), first at pH 7.5 and subsequently at pH 1.5 (adjusted with phosphoric acid); extraction at each pH was performed three times. The three corresponding ether extracts were pooled, dried over anhydrous Na₂SO₄, and stored at 4°C in glass bottles sealed with Teflon-coated screw caps until further analysis. Directly before analysis, each ether extract was evaporated to dryness, taken up in 1 ml of dichloromethane, and methylated using an etheral solution of diazomethane that was freshly prepared from Diazald (Sigma-Aldrich, Deisenhofen, Germany) according to standard procedures (Technical Bulletin AL113; Aldrich Chemical Co., Milwaukee, Wis.).

EPR spectroscopy. Cells for EPR analysis were harvested from six cultures (400 ml each) freshly grown with n-hexane or n-hexanoate. All steps were performed under strictly anoxic conditions either inside an anoxic chamber (Coy, Ann Arbor, Mich.) with a fresh oxygen-scavenging catalyst, or under gassing of tubes with pure N₂ (52). Cells from 2.4 liters of culture volume were centrifuged (7,000 × g, 20 min) and resuspended in anoxic water (10 ml) containing sodium dithionite (0.1 mM) to a final volume of approximately 5 ml. One part of this concentrated suspension was disrupted by passage through a French pressure cell (9.5-mm piston, 120 MPa). Removal of cell debris by centrifugation (20,000 × g, 20 min) yielded the crude extract. From this, another aliquot was used to sediment membranes (100,000 × g, 60 min); this supernatant is referred to as the soluble extract. Protein in lysed cells and extracts was quantified by the method of Bradford (12) with bovine serum albumin as the standard. Aliquots (each approximately 0.25 ml) from the cell suspension, the crude extract, and the soluble extract were transferred to anoxic EPR tubes, sealed under N₂, and immediately frozen and stored in liquid N₂.

Chemical synthesis of MPS and EBS. MPS and EBS as standard compounds were synthesized from succinic acid dimethyl ester and 2-hexanone or 3-hexanone, respectively. The reaction sequences involved Stobbe condensation of the carbonyl compounds with succinic acid dimethyl ester, dehydration yielding the unsaturated intermediate, and catalytic hydrogenation with Raney nickel, analogous to the synthesis of 3-hydroxybenzylsuccinate described elsewhere (35). This procedure of chemical synthesis yields racemic diastereomers of MPS and EBS. Methylation of the substituted succinic acids using a solution of diazomethane in diethyl ether yielded the respective dimethyl esters. The diastereomer mixtures of the dimethyl esters of MPS and EBS were isolated as colorless oily liquids from the reaction mixture by column chromatography on silica gel with a dichloromethane-methanol mixture (99:1). Structures were confirmed by ¹H and ¹³C nuclear magnetic resonance (NMR) spectra recorded on a Varian Gemini300 instrument (Varian, Darmstadt, Germany). Signals from (1-methylpentyl)succinic acid dimethyl ester (mixture of diastereomers) were as follows: ¹H NMR (300 MHz, CDCl₃, trimethylsilane [TMS]):  = 3.697 (s, 3H), 3.693 (s, 3H), 3.676 (s, 3H), 3.673 (s, 3H), 2.92-2.67 (m, 4H), 2.43-2.31 (m, 2H), 1.86-1.95 (m, 1H), 1.72-1.84 (m, 1H), 1.15-1.35 (m, 12H), 0.84-0.92 (m, 12H) ppm; ¹³C NMR (75 MHz, CDCl₃, TMS):  = 175.10, 174.68, 173.12, 172.96, 51.72, 51.68, 51.53, 46.03, 45.61, 35.03, 34.50, 34.01, 33.55, 33.27, 31.32, 29.39, 29.28, 22.67, 16.85, 16.19, and 13.98 ppm. Signals from (1-ethylbutyl)succinic acid dimethyl ester (mixture of diastereomers) were as follows: ¹H NMR (300 MHz, CDCl₃, TMS):  = 3.690 (s, 6H), 3.684 (s, 6H), 2.87-3.05 (m, 2H), 2.69-2.79 (m, 2H), 2.29-2.36 (m, 2H), 1.62-1.72 (m, 2H), 1.15-1.38 (m, 12H), 0.85-0.94 (m, 12H) ppm; ¹³C NMR (75 MHz, CDCl₃, TMS):  = 175.34, 173.18, 51.79, 51.70, 43.07, 41.21, 33.17, 32.97, 31.99, 31.78, 24.17, 23.70, 20.41, 20.26, 14.24, 14.19, 11.81, and 11.65 ppm.

Chemicals. Chemicals were of analytical grade and mostly obtained from Fluka (Deisenhofen, Germany) or Merck (Darmstadt, Germany). Purity of n-hexane was 99.5%. d₁₄-n-Hexane, 2,2,3,3-d₄-succinic acid, 2-hexanone, 3-hexanone, and succinic acid dimethyl ester were purchased from Sigma-Aldrich (Deisenhofen, Germany). [1-¹³C]n-Hexane and 2,3-d₂-fumaric acid were purchased from Campro Scientific (Berlin, Germany). 2,2,4,4,6,8,8-Heptamethylnonane was from Alfa/Johnson Matthey (Karlsruhe, Germany). Acids used as substrates (n-hexanoic acid, labeled fumaric acid, and labeled succinic acid) were titrated to yield aqueous solutions (1 M, pH 8) of sodium salts prior to addition to the medium. Diethyl ether of analytical grade was obtained from Riedel-de Haën (Seelze, Germany).

Calculation of free energies. G° values of reactions were calculated from G_f° values. The G_f° values (in kilojoules per mole) of the following species are given in the literature: fumarate² (aq), 604.2 (48); methane (g), 50.8 (48); n-hexane (lq), 4.3 (47); and toluene (lq), +114.2 (48). G_f° values of substituted succinates, which are not given in the literature, were calculated from increments using two different algorithms (34, 47). The G_f° values obtained (in kilojoules per mole) were as follows: methylsuccinate² (aq), 681.6 and 685.5; MPS² (aq), 647.3 and 644.0; and benzylsuccinate² (aq), 525.4 and 521.1. Respective G_f° values calculated via increments for succinate² (aq) as a test case were 687.5 and 690.0, compared to the literature value of 690.2 kJ/mol (48). The equilibrium constant was calculated as K = exp(G°/RT), where R is the gas constant and T is the absolute temperature.

	RESULTS

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Identification of MPS as a metabolite specifically formed from n-hexane. GC-MS analysis of ether extracts from cultures of strain HxN1 grown with n-hexane and subsequently pasteurized without access to air revealed 12 pronounced peaks of metabolites in the supernatant. These were not detectable in cultures grown with n-hexanoate. Peaks were only detectable if the culture (pH of about 7.5) was acidified before ether extraction, indicating that metabolites were carboxylates. No significant portions of metabolites were left in the cell pellet of the heat-treated culture.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Partial gas chromatograms of methylated extracts from heat-treated, acidified cultures of strain HxN1 after anaerobic growth with nitrate as the electron acceptor and with n-hexanoate (A) or n-hexane (B) as the only organic substrate. Both chromatograms were recorded at the same detection sensitivity. Mass spectrometric data from peaks are shown in Table 1; the mass spectrum of compound 3, which was essentially the same as of compound 2, is shown in Fig. 2A. Compounds 2 and 3 are interpreted as separable diastereomers of MPS dimethyl ester; possible diastereomers of compound 1, viz., of EBS dimethyl ester, were not separable on the gas chromatography column. Asterisks (*) indicate chiral carbon atoms; assignment of the absolute stereochemistry of diastereomers was not achieved in the present study.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Mass spectra of methylated MPS (diastereomer corresponding to peak 3 in Fig. 1B) from anaerobic cultures of strain HxN1 grown with nitrate as the electron acceptor and unlabeled and/or deuterium-labeled organic substrates. The following experiments were conducted (panel of recorded mass spectra indicated in parentheses): unlabeled n-hexane (A); mixture of unlabeled n-hexane and d14-n-hexane (A, B, C, and D); and unlabeled n-hexane and 2,3-d2-fumarate or 2,2,3,3-d4-succinate (A and C).

Labeling studies. To examine the formation of MPS in more detail, cultures were provided with labeled n-hexane or with labeled fumarate or succinate as possible precursors of the dicarboxylate moiety in MPS. Growth of cultures was not affected when unlabeled n-hexane was completely replaced by [1-¹³C]n-hexane. In contrast, when d₁₄-n-hexane was provided as the only organic substrate, no growth occurred within 2 weeks. Growth tests with mixtures of unlabeled n-hexane and d₁₄-n-hexane at various ratios revealed a 1:1 mixture (equal volumes) as the highest portion of deuterated n-hexane that still allowed growth; this ratio was therefore applied for labeling studies. Addition of 2,3-d₂-fumarate or 2,2,3,3-d₄-succinate to cultures growing with n-hexane did not impede growth; in contrast, these dicarboxylic acids even stimulated growth, because strain HxN1 uses them readily as carbon sources and electron donors (Behrends et al., unpublished).

Evidence for the presence of a glycyl radical. Dense cell suspensions, crude extract, and soluble extract from strain HxN1 grown on either n-hexane or n-hexanoate were studied by EPR spectroscopy. A characteristic intense EPR signal was observed in cells of n-hexane-grown cultures. (Fig. 3B). In crude and soluble extracts of n-hexane-grown cells, a signal with identical EPR parameters could also be observed, with intensity losses of 7 and 27%, respectively (data not shown). The g value (2.0032), line width, relaxational behavior (P_1/2 at 77 K is 0.05 mW), and strong hyperfine coupling to a single proton (1.5 ± 0.1 mT) are indicative of an organic radical centered at the -carbon of an amino acid. With n-hexanoate-grown cells, the signal indicative of such an organic radical was less than 4% of the signal from n-hexane-grown cells (Fig. 3A), and only a very weak nonhyperfine resolved isotropic signal at g = 2.004 with a line width of 1.7 ± 0.2 mT was present. The signal persisted in the crude and soluble extract, with similar amplitude and EPR parameters (not shown), and is tentatively assigned to a soluble flavoprotein in the semiquinone form. Quantification of the intense EPR signal by double integration and correction for the assumed presence of a flavin semiquinone background signal as in n-hexanoate-grown cells (only 10 to 20% of the double integral) indicated a concentration of the radical of 5 µM in the cell suspension. The protein content in the undiluted soluble extracts was 12.4 and 9.4 mg/ml for cells grown on n-hexanoate and n-hexane, respectively.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   EPR spectra of suspensions of cells of strain HxN1 grown anaerobically with n-hexanoate (A) and n-hexane (B) as the only organic substrates and nitrate as the electron acceptor. The relative amplitudes were normalized for equal concentrations of soluble protein. Conditions for recorded EPR spectra: 60 K; microwave power, 13 µW; modulation amplitude, 0.45 mT; microwave frequency, 9.46 GHz; modulation frequency, 100 kHz.

	DISCUSSION

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Supposed initial reaction of n-hexane. MPS was identified as a prominent metabolite in cultures of strain HxN1 grown anaerobically with n-hexane and nitrate. In parallel cultures grown on n-hexanoate, MPS was not detectable, indicating that this compound is specifically formed during n-hexane metabolism. The finding of the ¹³C label from [1-¹³C]n-hexane in the 1-methylpentyl side chain proves that this moiety originates from the n-alkane. This assumption is further confirmed by the observed preservation of 13 deuterium atoms from d₁₄-n-hexane in the 1-methylpentyl side chain of MPS. If n-hexane is the only organic substrate, the dicarboxylate moiety in MPS also has to be derived, at least in part, from the carbon of n-hexane; this transformation has to involve various metabolic steps that may include oxidation/reduction, cleavage, and carboxylation reactions. The origin of the dicarboxylate moiety of MPS is best explained if a C₄ dicarboxylic acid is assumed to be the direct precursor. Such an assumption agrees well with the hypothesized mechanistic relationship between the initial reactions of n-alkanes and of toluene in denitrifying bacteria (see the introduction). Toluene is activated by addition of the methyl group to fumarate as a cosubstrate, yielding benzylsuccinate (7, 9, 24, 31). In analogy to this, the formation of MPS can be explained by an addition of n-hexane with its subterminal carbon atom to fumarate (Fig. 4). Another metabolite, EBS, was identified in cultures of strain HxN1, and the amount of EBS extracted was 100-fold lower than that of MPS. It is true that a low pool size of a metabolite under nonequilibrium conditions in active cells does not necessarily indicate that the compound is of minor relevance. Nevertheless, there are hints that EBS is a by-product (formed by "accidental" reaction) rather than a true intermediate and that MPS is the principal product of the initial reaction of n-hexane degradation. Monocarboxylic organic acids (with fewer carbon atoms than MPS and EBS) detected in extracts from n-hexane-grown cultures and tentatively identified as possible subsequent metabolites contain methyl branches rather than ethyl branches (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, unpublished data).

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Proposed initial reaction of n-hexane with fumarate yielding MPS during anaerobic growth of denitrifying strain HxN1. Deuterium-labeled positions in a parallel experiment with 2,3-d2-fumarate added to an n-hexane-utilizing culture are indicated in grey.

Significance of the identified radical. The distinct EPR signal in n-hexane-grown cells and its virtual absence in n-hexanoate-grown cells of strain HxN1 provide evidence for the specific involvement of a radical in the anaerobic initial reaction of n-hexane. The observed signal is from a radical species which has a strong coupling to a single hydrogen atom. The average g value, line shape, and magnitude of the hyperfine coupling is atypical for radicals at carbon side chains of amino acids (viz., at amino acids other than glycine), flavin radicals, or sulfur-centered radicals. To our knowledge, there are presently no biochemically purified cell components other than glycyl radical enzymes which exhibit such an EPR signal. However, it cannot be excluded that a 1-methylpentyl radical rather than a glycyl radical is present in a protein that is specifically formed during growth of strain HxN1 on n-hexane. It is true that -carbon-centered radicals may occur at amino acids other than glycine (40); however, these would not have the observed strong hyperfine coupling to a single hydrogen atom. In anaerobic bacteria, signals like the present one have thus far only been observed in the case of glycyl radical enzymes (42). Such enzymes are pyruvate formate-lyase (27-29), anaerobic ribonucleotide reductase (36, 45, 46, 53), and the toluene-activating benzylsuccinate synthase (C. Leutwein, A. J. Pierik, and H. Heider, personal communication). Before the EPR measurement with benzylsuccinate synthase, evidence for the presence of a glycyl radical in benzylsuccinate synthase was provided by sequence similarity with other glycyl radical enzymes (17, 31), and polypeptide fragmentation upon exposure to oxygen (31). Hence, the EPR spectrum obtained from n-hexane-grown cells of strain HxN1 is in favor of the presence of a glycyl radical in this bacterium and further substantiates the assumed parallel between the initial reactions of n-alkanes and toluene in anaerobes. A small n-hexane-induced protein in strain HxN1 with N-terminal sequence similarity to the small subunit (BssC) of benzylsuccinate synthase (Behrends et al., unpublished) may be part of the assumed n-alkane-activating enzyme. Still, a definite assignment of the EPR signal to a glycyl radical would be premature at present and has to be substantiated by further experiments, such as EPR measurements upon growth with labeled glycine and purification of the radical-bearing species.

Energetic aspects of anaerobic initial reactions of hydrocarbons. Even if the mechanisms of the initial reactions of n-alkanes and toluene in anaerobes follow the same principle, the activation steps are not energetically equivalent. The energetic expenditure for homolytic (and also heterolytic) cleavage of a C-H bond is higher with an alkane than with the methyl group of toluene. The resulting alkyl radical cannot be stabilized like the benzyl radical with its -electron system. The C-H bond energies at the secondary carbon atom of an n-alkane and the methyl group of toluene are 401 and 368 kJ/molmol, respectively (33). However, these absolute values are not direct indicators of the actual activation energies of the hydrocarbons, because the transition state and its bond energies (such as that of transiently bonded H^· radical) at the enzyme are unknown. These values only indicate that the bond energy to be overcome in the case of n-hexane is 33 (= 401  368) kJ/mol higher than in the case of toluene. The bond energy to be overcome for the activation of an n-alkane at the primary (terminal) carbon (C-H bond energy, 419 kJ/mol) would be higher by as much as 51 kJ/mol in comparison to activation of toluene. This may explain why enzyme evolution has not favored n-hexane activation at the terminal carbon. A similar principle has been observed in the anaerobic synthesis of diabolic acid (15,16-dimethyltriacontanedioic acid), the only known reaction (19, 21) that resembles the anaerobic initial reaction of an alkane. Diabolic acid, a major lipid component in Butyrivibrio fibriosolvens, is formed by radical condensation of two n-hexadecanoic (palmitic) acid molecules at the subterminal carbon atom of their apolar residues. The reaction is assumed to proceed via simultaneous formation of a radical at each chain and hence differs in some detail from the suggested anaerobic alkane activation. Furthermore, radical formation in the synthesis of diabolic acid has been suggested to involve coenzyme B₁₂ (21), whereas the glycyl radicals investigated so far are generated with S-adenosylmethionine (20, 23). Among chemical reactions, the addition of alkanes (e.g., of propane at carbon-2) to alkenes via a radical mechanism (33) has some similarity to the suggested route of MPS formation. However, the more rapid chemical reaction is performed at 500°C and 10 MPa and yields an uncontrolled variety of products. In addition, an ionic mechanism for alkane-alkene addition catalyzed by very strong acids is known (33).