Proc. Natl. Acad. Sci. USA Vol. 81, pp. 6613-6617, November 1984 Biochemistry

Alkane biosynthesis by decarbonylation of catalyzed by a particulate preparation from Pisum sativum (plant wax/CO production/hydrocarbon) T. M. CHEESBROUGH AND P. E. KOLATTUKUDY* Institute of Biological Chemistry and Biochemistry/Biophysics Program, Washington State University. Pullman, WA 99164-6340 Communicated by P. K. Stumpf, June 29, 1984

ABSTRACT Mechanism of enzymatic conversion of a fat- MATERIALS AND METHODS ty acid to the corresponding by the loss of the carboxyl Materials. Omnifluor, [1-_4C]stearic acid, [U-3H]tetraco- carbon was investigated with particulate preparations from Pi- sanoic acid, [1-'4Cjsteroyl-CoA, and LiAl3H4 were from sum sativum. A heavy particulate preparation (sp. gr., 1.30 New England Nuclear. Sodium [1-14C]cyanide was from g/cm3) isolated by two density-gradient centrifugation steps ICN (Chemical and Radioisotopes Division). Pyridinium catalyzed conversion of octadecanal to heptadecane and CO. chlorochromate, meso-tetraphenylporphyrin, and RhCP Experiments with [1-3H,1_14C]octadecanal showed the stoichi- 3H2O were from Aldrich. LiAIH4, di-t-butylchlorophos- ometry of the reaction and retention of the aldehydic hydrogen phine, and Ru(CO)12 were from Alfa Products. The rhodium in the alkane during this enzymatic decarbonylation. This de- chelate was synthesized by the procedure of Monson (8). carbonylase showed an optimal pH of 7.0 and a Km of 35 ,uM Ruthenium dicarbonyl tetraphenylporphyrin [Ru(CO)2- for the . This enzyme was severely inhibited by metal (TTP)] was synthesized (9) and activated by substitution of ion chelators and showed no requirement for any cofactors. one CO with di-t-butyl-phosphine (10). The bright red crys- Microsomal preparations and the particulate fractions from tals of RuCO(TTP)[(C4H9)2P1 gave a spectrum identical to the first density-gradient step catalyzed acyl-CoA reduction to that reported (11). The oxidizing contaminants in Triton X- the corresponding aldehyde. Electron microscopic examina- 100 were removed as described (12). tion showed the presence of fragments of cell wall/cuticle but [1-14C]Octadecanal was synthesized from [1-'4C]octade- no vesicles in the decarbonylase preparation. It is concluded canol generated by LiAlH4 reduction of the corresponding that the aldehydes produced by the acyl-CoA reductase located acid using pyridinium chlorochromate, and the resulting al- in the endomembranes of the epidermal cells are converted to dehyde was purified as described (13). [2-14C]Octadecanal by the decarbonylase located in the cell wall/cuticle was synthesized by two cycles of nitrile elongation using a region. microscale adaptation of the method of Vederas et al. (14), followed by LiAlH4 reduction of the resulting acid and reoxi- Alkanes are widely distributed in the plant and animal king- dation of the alcohol to the aldehyde. [1-3H]Octadecanal was doms (1). Biological hydrocarbons usually have an odd num- synthesized by reduction of methyl octadecanoate with ber of carbon atoms, suggesting that they are derived by the LiAl3H4, followed by oxidation of the resulting alcohol with loss of one carbon atom from fatty acids with even numbers pyridinium chlorochromate as indicated above. [2-3H]Octa- of carbon atoms. Experiments with higher plant tissue slices decanoic acid was synthesized from 2-bromooctadecanoic strongly suggested that hydrocarbons are formed by chain acid generated by bromination of octadecanoic acid by the elongation of fatty acids followed by loss of the carboxyl Hell-Vollard-Zelensky reaction as described (15). The carbon, presumably by decarboxylation (2, 3). Subsequent methyl ester of the bromo acid was reduced with LiAl3H4 in work with insects (4) and mammals (5) supported this mech- tetrahydrofuran, and the resulting [1,2-3H]octadecanol was anism for alkane biosynthesis. Experiments with cell-free oxidized with CrO3 to [2-3H]octadecanoic acid. [U-3H]Te- preparations from pea leaves showed that oxygen and ascor- tracosanoyl-CoA was synthesized from the acid as described bate were required for the conversion of C32 fatty acid to (16). alkane and that metal ion chelators strongly inhibited alkane Enzyme Preparations. Pisum sativum (var. dark green per- synthesis (6). Subsequent studies showed that a major part fection) was grown in a growth room with a 220C day, 15'C of this alkane-generating activity was located in a crude mi- night, and a 15.5-hr photoperiod of 16,000 Ix. The apical crosomal fraction and that C18 to C32 fatty acids could serve meristem and its enclosing unopened leaflets were harvested as substrates for alkane formation (7). All of these substrates from 28- to 36-day-old plants. About 15 g of tissue was ho- gave rise to mainly alkanes containing two carbon atoms less mogenized three times for 10 sec each in an Omnimix with than the parent acid. Evidence was presented suggesting that 0.1 M potassium phosphate, pH 7.0/0.3 M sucrose. The ho- in vitro the aldehyde generated from the parent acid by the mogenate was filtered through two layers of cheesecloth and classical a-oxidation was the immediate precursor of the al- centrifuged for 20 min at 10,800 x g. The resulting superna- kane, whereas in vivo aldehydes generated by acyl-CoA re- tant was centrifuged at 165,000 x g, for 1 hr. The microsom- ductase might be the immediate precursor of alkanes. It was al pellet (P-2), resuspended in 24 ml of 0.1 M potassium suggested that the aldehyde might be decarborlylated to al- phosphate, pH 7.0/18% (wt/vol) sucrose, was layered on a kane. In the present paper, we describe the isolation of a discontinuous density gradient. Each tube, containing 6 ml particulate fraction devoid of a-oxidation activity, and we of sample layered on 7 ml of 25%, 6 ml of 30%, 8 ml of 40%, present direct experimental evidence for enzymatic decar- and 6 ml of 60% sucrose solutions in 0.1 M potassium phos- bonylation of an aldehyde to alkane. phate (pH 7.0), was centrifuged at 64,000 x g for 3 hr. The first 4 ml of 60% sucrose (G-1) collected from the bottom The publication costs of this article were defrayed in part by page charge was diluted 1:2 with 0.1 M potassium phosphate and centri- payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

6613 Downloaded by guest on September 28, 2021 6614 Biochemistry: Cheesbrough and Kolattukudy Proc. Natl. Acad Sci. USA 81 (1984)

fuged for 3.5 hr at 64,000 x g on a second discontinuous sucrose density gradient, which consisted of 8 ml of sample, 60 40 30 25 18 4 ml of 40% sucrose, and 3 ml of 60% sucrose. The first 2.5 ml of 60% sucrose collected from the bottom was diluted 1:2 1.2 Aldehyde fl with 0.1 M potassium phosphate and centrifuged at 165,000 n- x g for 1.5 hr. The pellet (G-2) was resuspended in 1 ml of 0.1 M potassium phosphate buffer (pH 7.0) and used as the 0.6 source of the decarbonylase enzyme. Electron Microscopic and Chemical Examination. The par- ticulate fraction, recovered by a 1:2 dilution and centrifuga- tion at x was 165,000 g for 30 min, fixed with 2% gluteralde- E 1.2 hyde and 2% OS04 and stained with lead citrate/uranyl ace- tate. The particulate fraction recovered from the gradients r- was depolymerized with LiAlH4, and the products were ex- 0.6 amined by combined gas-liquid chromatography and mass spectrometry as described (17). H= Decarbonylase Assays. The reactions were run in 16 x 125 mm test tubes with serum caps through which polypropy- lene cups were fitted. Each cup contained 1 mg of Rh- 0.03p- Cl[(C6H6)3P]3 on a strip of wetted filter paper. Each reaction mixture contained 0.1 M potassium phosphate (pH 7.0), 36 A.M octadecanal, and enzyme in 2.0 ml. The substrate solu- tion was prepared by sonicating 72 nmol of octadecanal in 0.c 0.5 ml of 0.1 M potassium phosphate (pH 7.0) with 0.1% )1, (vol/vol) purified Triton X-100. After incubating the mix- tures at 30'C for 45 min, 200 A.l of 2 M HCl was added, and 0 5 10 15 any bound CO was released by photolysis by placing a 22 W Fraction fluorescent light 4 cm away from the reaction mixture for 2 min. The filter paper trap containing the CO adduct was FIG. 1. Sucrose density-gradient fractionation of pea leaf micro- placed directly in scintillation fluid and assayed for 14C. The somes. The microsomal suspension was centrifuged on a discontinu- lipids were recovered from the reaction mixtures with ous sucrose density gradient; the interfaces of the 18%, 25%, 30c, CHCl3/CH30H (2:1; vol/vol), and the alkanes were isolated 40%, and 60%o sucrose layers are marked by arrows. Each fraction by thin-layer chromatography and assayed for radioactivity was assayed for a-oxidation activity with [1-14C]palmitic acid and as described (2). The alkane fraction was analyzed by radio for alkane synthesis with [U-3H]tetracosanoic acid. Aldehyde pro- duction in the latter assay was measured by thin-layer chromatogra- gas-liquid chromatography. phy. Trypsin Digestion of G-2. An aliquot (100 ,ul) of the enzyme was with 25 strokes in a 2-ml Ten preparation homogenized decanoic acid, the major cutin monomer (3), only in the frac- Broeck homogenizer with 1.1 ml of 0.1 M potassium phos- tions that contained decarbonylase activity including G-2, phate buffer, pH 7.0/20 ,ug of L-1-tosylamido-2-phenylethyl the final particulate preparation. chloromethyl -treated trypsin. After a 2-hr incubation Substrate for Alkane Synthesis. The relative efficiency of at 30°C, the digest was assayed for decarbonylase activity. production of alkanes from fatty acid, acyl-CoA, and alde- hyde changed with each step in the preparation of the hydro- RESULTS carbon-synthesizing particulate preparation (Table 1). The Isolation of an Alkane-Synthesizing Particulate Fraction. crude microsomal preparation (P-2) and the particulate prep- When the crude microsomal pellet was resuspended and cen- aration from the first gradient converted acyl-CoA 3-4 times trifuged on a discontinuous sucrose density gradient, a frac- as effectively as fatty acids into alkanes. This fraction also tion enriched in alkane-synthesizing activity was obtained. generated labeled octadecanal from octadecanoyl-CoA (data a-Oxidation activity was present at high levels in all but the not shown). The particulate fraction from the second gradi- 60% sucrose fractions (Fig. 1). These 60% fractions cata- ent readily converted aldehyde to alkane, whereas acyl-CoA lyzed a-oxidation 2 orders of magnitude slower than that ob- was a poor substrate and free acid was not converted into served with crude microsomes, but they readily catalyzed alkane. With the crude microsomal preparation, conversion alkane synthesis from fatty acids; aldehydes were, however, of aldehyde into alkane was barely detected. Presumably, generated by all fractions (Fig. 1). To minimize a-oxidation, which is the major reaction interfering with the alkane syn- Table 1. Relative efficiency of conversions of substrates into thesis assays, only the bottom 4 ml of the 60% sucrose (G-1) alkane and a-oxidation activity of the particulate preparations was used to study alkane synthesis. When the G-1 fraction from pea leaves was diluted and centrifuged on a second sucrose density gra- Hydrocarbon formation, the sucrose fraction of this con- dient, 60%o gradient (G-2) Particulate nmol/min per mg tained but no a-oxidation activ- a-Oxidation, alkane-synthesizing activity, fraction Fatty acid Acyl-CoA Aldehyde nmol/min per mg ity was present in these samples (Table 1). Thus, the double- gradient procedure yielded a fraction free of the major P-2 0.02 0.06 0.0002 1.56 (18.7) competing activity. The specific activity of alkane synthesis G-1 5.0 20.3 5.1 0.73 (0.023) increased from 0.01-0.02 nmol/min per mg of protein in G-2 0.0 0.60 24.0 0 crude extracts to 20-25 of in G-2. nmol/min per mg protein Assays with P-2 and G-1 used [n-3H]tetracosanoic acid or tetra- Electron microscopic examination of G-1 showed some vesi- cosanoyl-CoA prepared from it, whereas for assays with G-2, les that appeared to be plastids and fragments of cell wall/ [1-_4C]octadecanoic acid or the CoA ester prepared from it was cuticle. In the final preparation, G-2, only the latter could be used. [1-3H,1-_4C]Octadecanal was used where aldehyde is in- found (data not shown). Chemical examination of the partic- dicated. a-Oxidation was measured using [1-_4C]palmitic acid. Val- ulate fractions showed the presence of 10,16-dihydroxyhexa- ues in parentheses indicate total activity in nmol/min. Downloaded by guest on September 28, 2021 Biochemistry: Cheesbrough and Kolattukudy Proc. Natl. Acad. Sci. USA 81 (1984) 6615 Table 2. Isotopic ratios of alkane generated from dual-labeled dium and its chelates are known to form stable adducts with octadecanoic acid and octadecanal by a particulate fraction CO but have very low affinity for CO2 (18). Under our ex- from pea leaves perimental conditions, 50% of the 14Co generated by chemi- Alkane cal decarbonylation of [1-_4C]octadecanal was absorbed by Substrate (3H/14C) the Rh complex, but 14CO2 was not trapped by this reagent. When the particulate fraction (G-2) was incubated with [1- [2-3H,U-14C]Octadecanoic acid 1.00 3H,1-14C]octadecanal, the Rh complex trapped 14C, while [2-3H,2- 4C]Octadecanoic acid 0.96 hyamine hydroxide trapped no radioactivity. Therefore, CO [1-3H,2-'4C]Octadecanal 0.74 and not CO2 was generated from the aldehyde. When the The G-1 particulate fraction was used for the assay with the acid, same reaction mixture was extracted and the products were and G-2 was used for the assay with octadecanal. In each case, 36 subjected to thin-layer chromatography, 3H was found in the /,M substrate was used. alkane fraction. Radio gas-liquid chromatographic analysis of the alkane fraction showed that all of the 3H was con- the exogenous aldehyde was diverted into other products by tained in n-C17 alkane (Fig. 2). Incubation of G-2 with [1- competing reactions catalyzed by such preparations. As the 3H,2-14C]octadecanal (3H/ 4C, 1.0) gave C17 alkane with an particulate preparation was subjected to density-gradient pu- isotopic ratio of 0.74. These results rule out oxidation of the rification steps, aldehyde became an efficient precursor for substrate to the acid followed by decarboxylation as well as alkane and with the final preparation aldehyde was the pre- elongation and decarboxylation. ferred substrate. Under the standard assay conditions, an average of 1.28 Dual Isotope Labeling Experiments. Previously, a-hydroxy mol of alkane was produced per mol of CO trapped. Without acid was thought to be an intermediate in the conversion of a photolysis, the ratio of alkane to CO was even higher. Since fatty acid to alkane (4, 6), but the present results suggest that CO released during the reaction could be binding to some decarbonylation of the aldehyde generates alkane. To distin- guish between these two possibilities, dual-labeled sub- A strates were used, and the results are shown in Table 2. a- 100 Oxidation would be expected to cause loss of at least some of the 3H at the C-2 position of octadecanoic acid during al- kane formation. However, with either [2-3H,U-14C]- or [2- HC the 3H,2-14C]octadecanoic acid, the isotopic ratio of alkane ,75 - generated was identical with that of the substrate, showing \ that 3H at C-2 was not lost. In the case of [1-3H,2-14C]octade- canal, the bulk of 3H was retained in the alkane generated from it. These results strongly suggested that conversion of the acid to the alkane did not involve a-oxidation but proba- ,50 - bly involved aldehyde as the intermediate and that the decar- \~.co bonylation of the aldehyde took place with retention of 3H from C-1 of the aldehyde in the alkane. Direct Evidence for Decarbonylation. To detect and mea- sure the amount of CO produced during decarbonylation, we developed a trapping procedure using RhCl[(C6H6)3P]3. Rho-

0 0.~~~~~~~3

C17 C16 C18

0 5 10 15 Time, min Time, min FIG. 2. Radio gas-liquid chromatography of the alkane generated from [1-'4C,1-3H]octadecanal by the particulate fraction G-2. The FIG. 3. (A) Effect of pH on decarbonylase activity. The assays lower tracing shows the thermal conductivity detector response due were run in 0.1 M potassium phosphate (o and *), 0.1 M citrate to the co-injected n-alkanes indicated. A coiled 3.6-m stainless steel phosphate (e), or 0.1 M Tricine (A), using [1-3H,1-14C]octadecanal column of 2 mm i.d. packed with 15% FFAP on Chromosorb WHP as described, and the formation of both CO and alkane (HC) was (Gow-Mac, Bridgewater, NJ) held at 1900C was used, and the efflu- measured. (B) Time course of alkane and CO formations from [1- ent was passed through a Nuclear-Chicago radioactivity monitor. 3H,1-14C]octadecanal by the decarbonylase preparation. Downloaded by guest on September 28, 2021 6616 Biochemistry: Cheesbrough and Kolattukudy Proc. NatL Acad Sci. USA 81 (1984) centrations that inhibited alkane formation in crude prepara- tions (6, 20), showed little effect on decarbonylation. How- 1.8 ever, at a higher concentration (>5 mM), some inhibition of decarbonylation was observed. Metal ion chelating agents, E o HC such as EDTA and 4 (1 mM) o-phenanthroline (0.1 mM), se- S- fig 4 .Eu 1.2 verely inhibited decarbonylation. The enzyme was sensitive to trypsin treatment and was completely inactivated by freezing. - II, / I DISCUSSION , 0.6 When [1-14C,2-3H]octadecanal was incubated with the par- u ticulate preparation obtained by the two density-gradient -0.05 0 0.1 0.2 centrifugation steps, labeled heptadecane was the only al- A1 1/[SI, X10-6 kane generated. Obviously this process involved a loss of 0 20 40 60 one carbon, which was recovered as 14Co by the Rh com- Substrate, ,uM plex, which specifically binds CO. Although the amount of FIG. 4. Effect of the concentration [1-'4C,1-3H]octadecanal on CO released under the experimental conditions was too low the formation of CO and alkane by the decarbonylase preparation. to permit direct chemical identification, the above evidence strongly suggests that CO was the product of the reaction. components in the reaction mixture, it is not surprising that The amount of CO generated from [1-14C,2-3H]octadecanal the measured amount of CO was slightly less than the was nearly equal to the amount of alkane formed under al- amount of hydrocarbon generated. most all experimental conditions. The recovery of CO was Effect of pH, Time, and Concentration of Protein and Sub- slightly less than the stoichiometric amount, most probably strate on Decarbonylation. The decarbonylase showed activi- because some components present in the enzyme prepara- ty only in a fairly narrow range of pH (Fig. 3A). Little activi- tion bound a portion of the CO. This explanation is support- ty was seen below pH 6.0 and above pHI 8.0. Since a fairly ed by the observation that whenever the ratio of CO generat- sharp pH optimum at -7.0 was observed, all subsequent ex- ed to amount of protein in the reaction mixture was low, periments were done at this pH. At this optimal pH, the mea- recovery of CO showed the most drastic departure from stoi- sured amount of CO released was -80% of the amount of chiometric amounts. In spite of this technical difficulty, the alkane formed. Alkane formation and CO release from octa- stoichiometry of the products generated strongly suggested decanal showed near linear increases with time up to -30 that the enzyme preparation catalyzed the formation of 1 mol min of incubation, after which the rate began to slow down each of CO and heptadecane from each mol of octadecanal. (Fig. 3B). The amount of CO released relative to alkane Thus, the enzyme can be called a decarbonylase. An analo- formed remained constant with the time of incubation. The gous organic chemical reaction has been recently described, amount of alkane formed from octadecanal increased linear- in which case a Ru porphyrin complex catalyzes decarbonyl- ly with increasing protein concentration up to 4 ug/ml. The ation of aldehydes to alkanes (21). The present case is a bio- relative amount of CO released, when compared to alkane chemical analogue of this reaction. formed, decreased with increasing protein concentration, The sensitivity to boiling, freezing, and trypsin treatment suggesting that binding of CO to some components in the of the conversion of octadecanal to heptadecane and CO particulate preparation prevented quantitative trapping of suggested that the observed decarbonylation was an enzy- the CO generated. matic process. The time-course, protein concentration de- Alkane formation increased with increasing octadecanal pendence, pH dependence, and substrate concentration de- concentration, resulting in a typical Michaelis-Menten sub- pendence showed that the particulate preparation contained strate saturation pattern (Fig. 4). Double reciprocal plots an enzyme that catalyzed decarbonylation of octadecanal. were linear with an apparent Km of 35 uM for octadecanal. The mechanism of decarbonylation is not understood. Al- Release of CO did not show a typical saturation pattern in though ascorbate and oxygen were found to be required for that at very low substrate concentrations the expected maximal rates of alkane formation by crude cell-free prepa- amount of CO was not released, although at higher substrate rations (6), the present results show that decarbonylation per concentrations the CO release became normal. With the low se does not require ascorbate or oxygen. Most probably, amounts of CO generated at very low substrate concentra- generation of aldehyde from the substrate acid by a-oxida- tions, the trapping of CO by the Rh reagent was not effec- tion catalyzed by such crude preparations required these co- tive, probably because of competitive binding of CO by factors. In the nonenzymatic decarbonylation catalyzed by some components present in the enzyme preparation as not- Ru porphyrin complex, the metal ion is thought to partici- ed above. However, a double reciprocal plot, obtained with- pate in coordinating with CQ and the aldehydic hydrogen is out including the values acquired at very low substrate con- retained in the alkane formed (20). Inhibition of the present centration, was linear and showed the same intercept on the enzymatic decarbonylation by metal ion chelating agents v axis. strongly suggested that a metal ion is involved in this pro- Alkane formation from exogenous fatty acids catalyzed by cess. It is possible that the metal is part of a porphyrin com- previously described cell-free preparations (6, 7) was an indi- plex present in the preparation, although no direct evidence rect process involving a-oxidative formation of aldehydes. to support this possibility is available. In any case, the metal Therefore, the cofactor requirements observed with such ion requirement indicated by the inhibition by chelators and preparations cannot be considered valid for the final step the observed retention of the aldehydic hydrogen in the al- alone. Ascorbate, which was required for alkane formation kane produced by the present particulate preparation sup- in the crude preparations (6, 7), was not required for the port the conclusion that the mechanism of the enzymatic de- present decarbonylation. Reduced pyridine nucleotides (0.1 is probably analogous to that of the nonenzy- mM) showed little effect on decarbonylation. Imidazole (1 matic decarbonylation previously observed. mM), a known inhibitor of a-oxidation (19), inhibited the in- Subcellular localization of the decarbonylase is not under- direct alkane formation observed in the crude preparations stood. With the crude microsomal preparation, aldehyde (6, 7) but showed no effect on decarbonylation. Thiol com- was not a preferred substrate and the major alkane generated pounds, such as mercaptoethanol and dithioerythritol at con- contained two carbon atoms less than the substrate acid (6, Downloaded by guest on September 28, 2021 Biochemistry: Cheesbrough and Kolattukudy Proc. NatL Acad Sci. USA 81 (1984) 6617

7). Since this preparation was obviously a mixture of mem- part by Grant GM-18278 from the U.S. Public Health Service. This branes that indirectly generated alkanes from acids, one can- is Scientific Paper 6851, Project 2001 from the College of Agriculture University, Pullman, WA. not draw any conclusions about subcellular location from re- Research Center, Washington State sults obtained with it. After the first density-gradient step acyl-CoA became a preferred substrate for alkane synthesis, 1. Kolattukudy, P. E., ed. (1976) The Chemistry and Biochemis- and the alkane formed contained a substantial proportion of try ofNatural Waxes (Elsevier/North-Holland, Amsterdam). 2. Kolattukudy, P. E. (1967) Phytochemistry 6, 963-975. the homolog containing one carbon less than the acid. Obvi- 3. Kolattukudy, P. E. (1980) in The Biochemistry ofPlants. Lip- ously acyl-CoA reductase contained in this preparation must ids: Structure and Function, ed. Stumpf, P. K. (Academic, have generated aldehyde, which must have given the alkane New York), Vol. 4, pp. 571-645. by decarbonylation. In fact, formation of octadecanal from 4. Chu, A. J. & Bloomquist, G. J. (1980) Comp. Biochem. Physi- octadecanoyl-CoA was demonstrated with this preparation. ol. 68B, 313-317. The final density-gradient centrifugation gave a particulate 5. Cassange, C., Darriet, D. & Bourre, J. M. (1977) FEBS Lett. preparation that showed a strong preference for aldehyde as 82, 51-54. substrate for alkane formation and contained no detectable 6. Khan, A. A. & Kolattukudy, P. E. (1974) Biochem. Biophys. a-oxidation activity. The only reaction this relatively heavy Res. Commun. 61, 1379-1386. 7. Bognar, A. L., Paliyath, G., Rogers, L. & Kolattukudy, P. E. (1.30 g/cm3) particulate fraction catalyzed was decarbonyla- (1984) Arch. Biochem. Biophys., in press. tion. 8. Monson, R. S. (1971) Advanced Organic Synthesis: Methods Electron microscopic examination of the final decarbonyl- and Techniques (Academic, New York). ase preparation showed amorphous structures with no rec- 9. Cullen, D., Meyer, E., Srivastava, T. S. & Tsutsui, M. (1972) ognizable features characteristic of any subcellular organ- J. Chem. Soc. Chem. Commun., 584-585. elle. The fragments observed structurally resembled cell 10. Boschi, T., Bontempelli, G. & Mazzocchin, G.-A. (1979) In- wall/cuticle. In fact, chemical examination showed that the org. Chim. Acta 37, 155-160. heavy particulate fraction, but not the other fractions from 11. Domazetis, G., Tarpey, B., Dolphin, D. & James, B. R. (1980) the gradient, contained cutin. Thus, it is highly likely that the J. Chem. Soc. Chem. Commun., 939-940. matrix. 12. Ashani, U. & Catravas, G. N. (1980) Anal. Biochem. 109, 55- decarbonylase is associated with cell wall/cuticle 62. Therefore, it appears probable that the aldehydes generated 13. Agrawal, V. P. & Kolattukudy, P. E. (1978) Arch. Biochem. by the fatty acid chain elongation and reduction catalyzed by Biophys. 191, 452-465. the endoplasmic reticulum and/or other membranes are se- 14. Vederas, J. C., Graf, W., David, L. & Tamm, C. (1975) Helv. creted to the extracellular matrix where the decarbonylase is Chim. Acta 58, 1886-1898. located. Thus, the nature of alkane generated depends on the 15. Kolattukudy, P. E. (1970) Arch. Biochem. Biophys. 141, 381- aldehyde available for decarbonylation and not on the speci- 383. ficity of the decarbonylase. In fact, crude cell-free prepara- 16. Bishop, J. E. & Hajra, A. K. (1980) Anal. Biochem. 106, 344- tions generated alkanes from C16 to C32 fatty acids with no 350. of 17. Walton, T. J. & Kolattukudy, P. E. (1972) Biochemistry 11, significant chain-length specificity (7). The high degree 1885-1897. specificity observed in vivo probably arises from the speci- 18. Orgel, L. E. (1966) Introduction to Transition Metal Chemistry ficity of the elongating system that provides the fatty acids (Butler & Tanner, London). for reduction and decarbonylation. Until the decarbonylase 19. Martin, R. 0. & Stumpf, P. K. (1959) J. Biol. Chem. 234, is solubilized and characterized, the nature of the enzyme 2548-2554. and the mechanism of decarbonylation cannot be elucidated. 20. Buckner, J. S. & Kolattukudy, P. E. (1973) Arch. Biochem. Biophys. 156, 34-45. We thank Linda Rogers for conducting some of the preliminary 21. Domazetis, G., James, B. R., Tarpey, B. & Dolphin, D. (1981) work, Sally Combelic for raising the plants, and Jim Huber for as- in Catalytic Activation of Carbon Monoxide, ed. Ford, P. C. sistance with the electron microscopy. This work was supported in (Am. Chem. Soc., Washington, DC), pp. 243-252. Downloaded by guest on September 28, 2021