Báo cáo Y học: Mechanism of 1,4-dehydrogenation catalyzed by a fatty acid (1,4)-desaturase of Calendula officinalis pptx

Mechanism of 1,4-dehydrogenation catalyzed by a fatty acid (1,4)-desaturase of Calendula officinalis Darwin W. Reed 1 , Christopher K. Savile 2 , Xiao Qiu 1 , Peter H. Buist 2 and Patrick S. Covello 1 1 Plant Biotechnology Institute, Saskatoon, SK, Canada; 2 Department of Chemistry, Carleton University, Ottawa, Ontario, Canada The mechanism by which the fatty acid (1,4)-desaturase of Calendula officinalis produces calendic acid from linoleic acid has been probed through the use of kinetic isotope effect (KIE) measurements. This was accomplished by incubating appropriate mixtures of linoleate and regiospecifically dideuterated isotopomers with a strain of Saccharomyces cerevisiae expressing a functional (1,4)-desaturase. GC-MS analysis of methyl calendate obtained in these experiments showed that the oxidation of linoleate occurs in two discrete steps since the cleavage of the C11-H bond is very sensitive to isotopic substitution (k H /k D ¼ 5.7 ± 1.0) while no isotope effect (k H /k D ¼ 1.0 ± 0.1) was observed for the C8-H bond breaking step. These data indicate that calendic acid is pro- duced via initial H-atom abstraction at C11 of a linoleoyl substrate and supports the hypothesis that this transforma- tion represents a regiochemical variation of the more com- mon C12-initiated D 12 desaturation process. Keywords: desaturase; kinetic isotope effect; conjugated fatty acid; deuterium labelling; Calendula. The D 12 -oleate desaturase (FAD2) family of enzymes are membrane-bound nonheme iron-containing proteins that carry out a fascinating array of related oxidative transfor- mations [1,2]. The prototypical reaction features the intro- duction of a cis-double bond at the 12,13-position of an oleoyl substrate – a ubiquitous biosynthetic reaction of higher plants [3,4] (Fig. 1A). Species-specific mechanistic variations of this process include 12-hydroxylation of oleate (Ricinus communis) [5] and 12,13-epoxidation (Crepis palaestina) [2] or 12,13-acetylenation (Crepis alpina)[2]of linoleate. Recently, it has been shown that the production of conjugated trienoic acids, such as calendic acid from a linoleate precursor are also carried out by FAD2 variants [6–8](Fig. 1B). The latter reaction is particularly noteworthy given the current interest in conjugated fatty acids with respect to their role in human nutrition [9] as well as commercial applications [10]. As part of ongoing research into the structure–function relationships of FAD2 type enzymes, a closer examination of calendate formation is clearly warranted. Early labelling experiments using marigold seed homogenates and labelled linoleate precursors demonstrated that calendic acid is produced by an apparent (1,4)-dehydrogenation process whereby a linoleoyl substrate loses one hydrogen from C8 and C11, respectively [11]. No oxygenated intermediates were detected. These results as well as related substrate specificity data [6] point to a mechanism which is analogous to that proposed for the more common (1,2)-dehydrogen- ation reactions of fatty acid desaturases (Fig. 2). The mechanistic model [12] for the latter process features an initial, energetically difficult hydrogen abstraction step, which generates a very short-lived, carbon-centered radical intermediate, or its iron-bound equivalent (not shown). This species collapses rapidly to give an unsaturated product by what is formally a second hydrogen abstraction, although a one electron oxidation/proton removal sequence cannot be rigorously excluded at this time. The stepwise nature of this transformation is supported by kinetic isotope effect (KIE) studies of several membrane-bound fatty acid desaturases. In all cases examined, one C-H cleavage was found to be subject to a large primary deuterium kinetic isotope effect while the second C-H bond rupture was insensitive to isotopic substitution [12–22]. This pattern of KIEs is precisely what one would expect for a disproportionation mechanism [23] of the type showed in Fig. 2 and the data were used to pinpoint the site of the initial oxidative attack (ÔcryptoregiochemistryÕ) for these systems. In several cases, additional independent evidence is available to support the cryptoregiochemical assignments. That is, the location of the putative diiron oxidant relative to substrate can be ascertained by inducing the desaturase to behave as a regioselective oxygenase through modifications of substrate or enzyme [19,20,24–26]. The availability of a convenient yeast expression system for Fac2 –aCalendula officinalis gene encoding the (1,4)- desaturase involved in calendic acid production [6,8] offered a unique opportunity to study the mechanism of this reaction using KIE methodology. Specifically, we wished to correlate the site of initial oxidation for this process with that determined for D 12 -desaturation [13]. We report here, the results of our collaborative investigation. Correspondence to P. H. Buist, Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada, K1S 5B6. Fax: + 1 613 5203749 or 3830, Tel.: + 1 613 5202600 Ext. 3643, E-mail: pbuist@ccs.carleton.ca or P. S. Covello, National Research Council, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9. Fax: + 1 306 9754839, Tel.: + 1 306 9755269; E-mail: Patrick.Covello@nrc.ca Abbreviations: KIE, kinetic isotope effect. Definition: The term (1,4)-desaturase denotes an enzyme that converts an isolated carbon-carbon double bond in a fatty acid into two conjugated double bonds by what is formally a 1,4-dehydrogenation reaction. Such enzymes have also been termed conjugases [7]. (Received 23 July 2002, accepted 28 August 2002) Eur. J. Biochem. 269, 5024–5029 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03209.x EXPERIMENTAL PROCEDURES Materials Methyl linoleate (> 99%) was purchased from Nu-Chek- Prep, Inc. The two regiospecifically dideuterated methyl linoleates ([8,8 2 H 2 ]-1,11,11- 2 H 2 ]-1) required for the KIE study were prepared by routes which were very similar to those reported previously for the synthesis of the corres- ponding chiral monodeutero analogues [27]. Thus, the tosylate of 8-hydroxy-[8,8 2 H 2 ]octanoic acid was reacted with lithium acetylide-ethylenediamine complex to give [8,8 2 H 2 ]dec-9-ynoic acid which was in turn coupled with the tosylate of 2-octyn-1-ol to give [8,8 2 H 2 ]-1 after semihydro- genation and methyl esterification of the intermediate diyne. In a similar manner, dec-9-ynoic acid was C-alkylated with the tosylate of [1,1- 2 H 2 ]-2-octyn-1-ol to give [11,11- 2 H 2 ]-1 after semihydrogenation and methyl esterification. The overall yields of [8,8 2 H 2 ]-1 and [11,11- 2 H 2 ]-1 obtained via these procedures was 5% and 14%, respectively. Purifica- tion of substrates was carried out by flash chromatography (silica gel, 0.5% v/v ethyl acetate/hexane) and HPLC fractionation as previously described [16]. GC-MS analysis [16] of the final deuterated products revealed that each isotopomer consisted essentially entirely of dideuterated species (m/z 296; 294 for nondeuterated analogue). 1 Hand 13 C NMR analysis confirmed the position of the two deuterium atoms for each isotopomer as indicated by the presence/absence of the diagnostic bisallylic signals at d 2.77 p.p.m. ( 1 H) and 25.67 p.p.m. ( 13 C) for [11,11- 2 H 2 ]-1, respectively, and an approximately two-fold attenuation of the overlapping allylic signals (C-8,C-13) at d 2.02 p.p.m. ( 1 H) and 27.25 p.p.m. ( 13 C) for [8,8 2 H 2 ]-1. Incubation experiments For characterization of the Calendula fatty acid conjugase, the yeast strain DTY10-a2 (MATa, fas2D::LEU2, can1- 100, ura3-1, ade2-1, his3-11, his3-15) [28] was transformed with a plasmid (pYJ) comprised of the pYES2 vector (Invitrogen) and the Fac2 gene from Calendula officinalis [6] to give the strain pYJ/DTY10-a2. While not a requirement for the experiments described, this strain harbours a mutation in fas2, rendering it unable to synthesize fatty acids de novo. Consequently, it was routinely grown in minimal media containing galactose [29],  0.1 m M pentadecanoic acid and 0.1% Tergitol type NP-40 (a polymer of ethylene oxide and p-nonylphenol). For fatty acid feeding experiments, cultures were supple- mented with  0.3 m M substrate fatty acids and incubated at 20 °C for three days followed by 15 °C for three days. Yeast cultures (D 600  4) were pelleted by centrifugation (4000 g, 10 min) and pellets were washed with 10 mL 1% Tergitol solution and 2 · 10 mL H 2 O prior to lipid extraction. Analytical procedures For fatty acid analysis, yeast pellets were saponified by adding 2 mL 10% KOH/methanol and heating at 80 °Cfor 2 h. The mixture was then cooled and pre-extracted with 2 · 2 mL hexane to remove nonsaponifiable lipids. The reaction mixture was then neutralized with 50% acetic acid to  pH 5 and the fatty acids were extracted with 2 · 2mL hexane. The hexane was removed under a nitrogen stream and the mixture, including the conjugated fatty acids, was esterifiedwith2mL1%H 2 SO 4 in methanol at 50 °Cfor1 h (This methylation method has been found to be most suitable for conjugated fatty acid ester analysis; W. Christie, Mylnefield Research Services Ltd., Dundee, Scotland, personal communication.) The cooled mixture was extrac- tedwith2· 2 mL hexane. The pooled hexane was washed with 2 mL H 2 O and concentrated under N 2 for HPLC purification, GC or GC-MS analysis. GC-MS analysis of yeast lipids was performed using a Fisons VG TRIO 2000 mass spectrometer (VG Analytical, UK) controlled by Masslynx version 2.0 software, coupled to a GC 8000 Series gas chromatograph as previously described [16] except that a narrow EI + scan range of 285– 305 m/z was used. A representative mass spectrum of biosynthetic methyl calendate is shown in Fig. 3. RESULTS AND DISCUSSION Our methodology for determining the intermolecular primary deuterium KIE on each C-H cleavage step in fatty acid desaturation reactions involves GC-MS analysis of olefinic products derived from direct competition between nondeuterated substrate and the appropriate regiospecifi- cally dideuterated (-C 2 H 2 -) fatty acid. As has been pointed out on a previous occasion [13], the magnitude of the primary deuterium KIE determined in this manner must be regarded as an estimate since the observed value may incorporate a small (< 10%) a-secondary isotope effect [40]. In addition, partial masking of the ÔintrinsicÕ KIE by Fig. 1. Biosynthesis of linoleate and calendate catalyzed by (1,2) and (1,4)-desaturases, respectively. OPL, phospholipid ester; X, undefined headgroup. Fig. 2. Generic mechanistic scheme showing the stepwise removal of hydrogens in fatty acid (1,2)-desaturation. Structure of the putative diiron oxidizing species is speculative. Ó FEBS 2002 Calendic acid biosynthesis (Eur. J. Biochem. 269) 5025 other enzymic steps in the catalytic cycle such as substrate binding may also be occurring [35]. None of these considerations affect the conclusions reached in this paper. The use of a competitive rather than a noncompetitive experimental design has allowed KIE determinations to be carried out for both in vitro and in vivo desaturase systems. The results have correlated well with KIE data obtained by other methods [30–34]. Our methodology dictates that interference by endogenous d 0 -substrate, if present, must be eliminated: this has been accomplished previously through the use of unnatural chain-shortened substrates or ana- logues bearing a remote ÔthiaÕ- or deuterium mass label. Such measures proved unnecessary in the case of the linoleate-calendate reaction since the host yeast system used for this purpose does not biosynthesize the relevant substrate. Optimal incubation conditions for our KIE studies were set up in a preliminary experiment: methyl linoleate 1 (100 mgÆL )1 ) was administered to cultures (50 mL) of the pYJ/DTY10a2 strain of S. cerevisiae incubated at 20 °Cfor 3 days to permit relatively rapid growth and then at 15 °C for a further 3 days to reach saturation at a temperature which has been found to give better substrate conversion rates. The cells were harvested by centrifugation and the lipids were isolated via a hydrolysis/methylation sequence known to be suitable for conjugated fatty acid esters (10% w/v KOH/CH 3 OH, 80 °C, 2 h; neutralization to  pH 5 with acetic acid, hexane extraction, 1% w/v H 2 SO 4 / CH 3 OH, 50 °C, 1 h). Analysis of the fatty acids as methyl esters by GC-MS revealed that exogenous linoleate had been converted to calendate to the extent of  1% of total cellular fatty acids, a result similar to that observed previously [6,8]. Control experiments previously indicated that the production of 2 was dependent on expression of the FAC2 enzyme. The two regiospecifically dideuterated linoleates ([8,8- 2 H 2 ]-1, [11,11- 2 H 2 ]-1) required for the KIE study (Fig. 4) were prepared via established synthetic routes (See Experimental section). A mixture of each deuterated material with its nondeuterated parent (1 mg) was admini- stered to growing cultures (10 mL) of the S. cerevisiae transformant (pYJ/DTY10-a2) using conditions identical to that of the trial experiment. The deuterium content of the olefinic fatty acid methyl esters in the cellular lipid extract was assessed by GC-MS as described in the Experimental section. The d 2 /d 0 ratio of the linoleate isotopomers found in the cells was essentially identical to that of the starting material in both incubations, as is required for these types of competitive KIE measurements [35]. No loss of label due to reversible exchange of deuterated linoleate at C-8 or C-11 could be detected. Mass spectral analysis of the calendate fraction revealed that in both incubations, this material consisted entirely of a d 0 /d 1 mixture indicating a loss of one deuterium from the d 2 -substrate as expected. Product kinetic isotope effects (k H /k D ) were calculated using the ratio: [% d 0 (product)/% d 1 (product)]/[% d 0 (substrate)/% d 2 (substrate)] and this analysis indicated the presence of a large primary deuterium isotope effect (5.7 ± 1.0, average of four experiments) for the carbon-hydrogen bond clea- vage at C11 while the C8-H bond breaking step was shown to be essentially insensitive to deuterium substitution (KIE ¼ 1.0 ± 0.1, average of four experiments) (Table 1). According to our mechanism (Fig. 2), these results demon- strate that calendate production is initiated by an energeti- cally difficult and hence isotopically sensitive hydrogen abstraction at C11 and completed by a second facile and kinetically unimportant hydrogen abstraction at C8. The fast formation of an allylic radical at C8 followed by rate- determining hydrogen abstracton at C11 cannot be rigor- ously excluded but seems far less likely given the intrinsically high energy content of radical intermediates relative to product. Some decades ago, Morris and Marshall [36] speculated that conjugated trienoic fatty acids are produced in plants from linoleic acid via an allylic radical intermediate. Crombie and coworkers [11] provided evidence that calen- dic acid was indeed biosynthesized from linoleic acid via removal of hydrogens at C8 and C11. More recently, it was suggested that C8 might be the site of initial oxidation for this process based on a comparison with the putative site of initial attack catalyzed by a soluble plant D 9 desaturase [37]. However our results clearly demonstrate that calendate production is in fact initiated at C11 as might be expected for a process which is catalyzed by a homolog of FAD2 – an enzyme which initiates the conversion of oleate to linoleate at C12 [12]. Thus, the switch between 1,2 and 1,4- dehydrogenation could conceivably be controlled by a fairly small change in oxidant position relative to substrates which both adopt a conformation allowing syn removal of Fig. 4. Isotopomers of 1 used to probe the kinetic isotope effects on the fatty acid (1,4)-desaturase reaction involved in calendate biosynthesis. Fig. 3. Mass spectrum of biosynthetic methyl calendate. Arrow indi- cates the molecular ion cluster used to calculate the isotopic content of deuterated samples. 5026 D. W. Reed et al. (Eur. J. Biochem. 269) Ó FEBS 2002 two proximal hydrogens (H-H distance in both cases  2.5 A ˚ ). This model (Fig. 5) can be tested by determining the stereochemistry of H-removal for calendate formation using chiral monodeutero probes [27] and comparing this result with the known pro-R enantioselectivity at C12,13 observed for D 12 -desaturation [38]. Further evidence for the close relationship between 1,2 and 1,4-dehydrogenation has been obtained recently for a Spodoptera littoralis desaturating system which converts 11(Z)- tetradecenoate to 10(E),12(E)-tetradecadienoate by initial H-abstraction at C10 and 11(E)-tetradecenoate to 9(Z),11(E)-tetradecadienoate by initial oxidative attack at C9 [39]. Whether these two transformations are catalyzed by separate enzymes in this case remains to be determined. In summary, the cryptic site of initial oxidation for an important plant fatty acid conjugase-mediated reaction has been determined to be at the carbon furthest from C-1. In contrast, all other desaturase-catalyzed oxidations studied to date are initiated at the carbon closest to the acyl headgroup. Thus it would be interesting to apply our KIE methodology to the study of related FAD2-like enzymes [7] involved in the formation of a-eleostearic acid [9(Z),11(E),13(E)-octadecatrienoic acid] and a-parinaric acid [9(Z),11(E),13(E),15(Z)-octadecatetraenoic acid] from linoleic acid. In so doing, we would hope to correlate the various regioselectivities observed for this important set of catalysts with the geometric relationship between oxidant and substrate. Note added in proof: Recent site-directed mutagenesis experiments using a D 12 desaturase/hydroxylase system have validated the mechanistic paradigm underlying our crypto- regiochemical determinations [41]. ACKNOWLEDGEMENTS We wish to thank the National Science and Engineering Research Council (NSERC) for financial support of the synthetic work performed at Carleton University (PHB), Steve Ambrose for perform- ing the GC-MS analysis, Charles Martin for providing the yeast strain DTY-10a2 and Michele Loewen and Robert Sasata for reviewing the manuscript. REFERENCES 1. Shanklin, J. & Cahoon, E.B. (1998) Desaturation and related modifications of fatty acids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 611–641. Fig. 5. Mechanistic model showing the rela- tionship between oxidant position in D 12 desat- uration and the (1,4)-desaturase reaction leading to the formation of a conjugated 8,10- diene system. Table 1. Intermolecular isotopic discrimination by the C. officinalis (1,4)-desaturase in the 1,4-dehydrogenation of [8,8 2 H 2 ]-linoleate and [11,11- 2 H 2 ]- linoleate. Isotopic Ratio a Substrates b Products d 0 :8d 2 d 0 : 11d 2 d 0 :8d 1 d 0 : 11d 1 KIE 1.07 6.99 6.5 1.00 4.41 4.4 1.09 5.65 5.2 1.04 6.80 6.5 (5.7 ± 1.0) c 1.95 1.93 0.99 2.08 2.04 0.98 2.15 2.05 0.95 1.72 2.04 1.19 (1.03 ± 0.11) c a The isotopic ratio of each species is given as an average value based on four GC-MS runs. d 0 ,d 1 ,d 2 refer to undeuterated, monodeuterated and dideuterated species, respectively. b The isotopic ratios of the substrates were determined by mass spectral examination of the linoleate isotopomers isolated from the cellular pool. These values are similar (within experimental error) to those obtained for the two starting mixtures prior to incubation: 1.02 (d 0 : 11d 2 ); 1.92 (d 0 :8d 2 ). c The average KIE (four incubations) ± standard deviation. Ó FEBS 2002 Calendic acid biosynthesis (Eur. J. Biochem. 269) 5027 2. Lee, M., Lenman, M., Banas, A., Bafor, M., Singh, S., Schweizer, M., Nilsson, R., Liljenberg, C., Dahlqvist, A., Gummeson, P O., Sjo ¨ dahl, S., Green, A. & Stymne, S. (1998) Identification of non- heme diiron proteins that catalyze triple bond and epoxy group formation. Science 280, 915–918. 3. Okuley, J., Lightner, J., Feldmann, K., Yadav, N., Lark, E. & Browse, J. (1994) Arabidopsis fad2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell 6,147– 158. 4. Heinz, E. (1993) Biosynthesis of polyunsaturated fatty acids. In: Lipid Metabolism in Plants (Moore, T.S. Jr, ed.), pp. 33–89. CRC Press, Boca Raton. 5. van de Loo, F.J., Broun, P., Turner, S. & Somerville, C. (1995) An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog. Proc. Natl Acad. Sci. USA 92, 6743–6747. 6. Qiu, X., Reed, D.W., Hong, H., MacKenzie, S.L. & Covello, P.S. (2001) Identification and analysis of a gene from Calendula offi- cinalis encoding a fatty acid conjugase. Plant Physiol. 125,847– 855. 7. Cahoon, E.B., Carlson, T.J., Ripp, K.G., Schweiger, B.J., Cook, G.A., Hall, S.E. & Kinney, A.J. (1999) Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos. Proc. Natl Acad. Sci. USA 96, 12935–12940. 8. Cahoon, E.B., Ripp, K.G., Hall, S.E. & Kinney, A.J. (2001) Formation of conjugated D 8 ,D 10 -double bonds by D 12 -oleic- acid desaturase-related enzymes: biosynthetic origin of calendic acid. J. Biol. Chem. 276, 2637–2643. 9. Pariza, M.W., Park, Y. & Cook, M.E. (2001) The biologically active isomers of conjugated linoleic acid. Prog. Lipid Res. 40, 283–298. 10. Derksen, J.T.P., Cuperus, F.P. & Kolster, P. (1995) Paints and coatings from renewable resources. Ind. Crops Prod. 3, 225–236. 11. Crombie, L. & Holloway, S.J. (1985) The biosynthesis of calendic acid, octadeca-(8E,10E,12IX)-trienoic acid, by developing marigold seeds: origins of (E,E,Z)and(Z,E,Z) conjugated triene acids in higher plants. J. Chem. Soc. Perkin Trans. I, 2425– 2434. 12. Buist, P.H. & Behrouzian, B. (1998) Deciphering the cryptor- egiochemistry of oleate D 12 desaturase: a kinetic isotope effect study. J. Am. Chem. Soc. 120, 871–876. 13. Buist, P.H. & Behrouzian, B. (1996) Use of deuterium kinetic isotope effects to probe the cryptoregiochemistry of D 9 desatura- tion. J. Am. Chem. Soc. 118, 6295–6296. 14. Pinilla, A., Camps, F. & Fabrias, G. (1999) Cryptoregiochemistry of the D 11 -myristoyl-CoA desaturase involved in the biosynthesis of Spodoptera littoralis sex pheromone. Biochemistry 38, 15272– 15277. 15. Abad, J.L., Camps, F. & Fabria ` s, G. (2000) Is hydrogen tun- neling involved in acylCoA desaturase reactions? The case of a D 9 desaturase that transforms (E)-11-tetradecenoic acid into Z and E-9,11-tetradecadienoic acid. Angew. Chem. Int. Ed. 39, 3279– 3281. 16. Meesapyodsuk, D., Reed, D.W., Savile, C.K., Buist, P.H., Ambrose, S.J. & Covello, P.S. (2000) Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase (FAT-1) expressed in Saccharomyces cerevisiae. Biochemistry 39, 11948–11954. 17. Meesapyodsuk, D., Reed, D.W., Cheevadhanarak, S., Deshnium, P. & Covello, P.S. (2001) Probing the mechanism of a cyano- bacterial D 9 fatty acid desaturase from Spirulina platensis C1 (Arthrospira sp. PCC 9438). Comp Biochem. Physiol. B Biochem. Mol. Biol. 129, 831–835. 18. Savile, C.K., Reed, D.W., Meesapyodsuk, D., Covello, P.S. & Buist, P.H. (2001) Cryptoregiochemistry of a Brassica napus fatty acid desaturase (FAD3): a kinetic isotope effect study. J. Chem. Soc., Perkins Trans. 1, 1116–1121. 19. Savile, C.K., Fabrias, G. & Buist, P.H. (2001) Dihydroceramide D 4 desaturase initiates substrate oxidation at C- 4. J. Am. Chem. Soc. 123, 4382–4385. 20. Fauconnot, L. & Buist, P.H. (2001) c-Linolenic acid biosynthesis: cryptoregiochemistry of D 6 desaturation. J. Org. Chem. 66, 1210– 1215. 21. Fauconnot, L. & Buist, P.H. (2001) Cryptoregiochemical analysis of an unusual bacterial desaturation. Bioorg.Med.Chem.Lett.11, 2879–2881. 22. Behrouzian, B., Fauconnot, L., Daligault, F., Nugier-Chauvin, C., Patin, H. & Buist, P.H. (2001) Mechanism of fatty acid desa- turation in the green alga Chlorella vulgaris. Eur. J. Biochem. 268, 3545–3549. 23. Cook, G.K. & Mayer, J.M. (1994) C-H bond activation by metal- oxo species: oxidation of cyclohexane by chromyl chloride. J. Am. Chem. Soc. 116, 1855–1868. 24. Buist, P.H. & Marecak, D.M. (1992) Stereochemical analysis of sulfoxides obtained by diverted desaturation. J. Am. Chem. Soc. 114, 5073–5080. 25. Buist, P.H., Alexopoulos, K.A., Behrouzian, B., Dawson, B. & Black, B. (1997) Synthesis and desaturation of monofluorinated fatty acids. J. Chem. Soc. Perkin Trans. 1, 2617–2624. 26. Broun, P., Shanklin, J., Whittle, E. & Somerville, C. (1998) Catalyic plasticity of fatty acid modification enzymes underlying chemical diversity of plant lipids. Science 282, 1315– 1317. 27. Crombie, L. & Heavers, A.D. (1992) Synthesis of linoleic acid with chiral isotopic labelling at a flanking and a medial allylic methy- lene: the (8R,9Z,12Z)-[8- 2 H] and (11R,9Z,12Z)-[11- 2 H]-stereo- isomers, and (Z)-[2,2- 2 H 2 ]-non-3-enal. J. Chem. Soc., Perkins Trans. 1929–1937. 28. Toke, D.A. & Martin, C.E. (1996) Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevi- siae. J. Biol. Chem. 271, 18413–18422. 29. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seid- man, J.G., Smith, J.A., Struhl, K., Albright, L.M., Coen, D.M. & Varki, A., eds (1995) Current Protocols in Molecular Biology. John Wiley and Sons, New York. 30. Schroepfer, G.J. & Bloch, K. (1965) The stereospecific conversion of stearic acid to oleic acid. J. Biol. Chem. 240, 54–63. 31. Baenziger, J.E., Smith, I.C.P. & Hill, R.J. (1990) Biosynthesis and characterization of a series of deuterated cis, cis-octadeca-6,9- dienoic acids. J. Chem. Phys. Lipids 54, 17–23. 32. Rahier, A. (2001) Deuterated Delta (7)-cholestenol analogues as mechanistic probes for wild-type and mutated delta (7)-sterol-C5 (6)-desaturase. Biochemistry 40, 256–267. 33. Rettie, A.E., Boberg, M., Reetenmeier, A.W. & Baillie, T.A. (1988) Cytochrome P-450-catalyzed desaturation of valproic acid in vitro: species differences, induction effects, and mechanistic studies. J. Biol. Chem. 263, 13733–13738. 34. Enoch, H.G., Catala ´ , A. & Strittmatter, P. (1976) Mechanism of rat liver microsomal stearyl-CoA desaturase. Studies of the sub- strate specificity, enzyme–substrate interactions, and the function of lipid. J. Biol. Chem. 251, 5095–5103. 35. Northrop, D.B. (1981) The expression of isotope effects on enzyme-catalyzed reactions. Annu.Rev.Biochem.50, 103–131. 36. Morris, L.J. & Marshall, M.O. (1966) Occurence of cis,trans- linoleic acid in seed oils. Chem. Ind., 1493. 37. Fritsche,K.,Hornung,E.,Peitzsch,N.,Renz,A.&Feussner,I. (1999) Isolation and characterization of a calendic acid producing (8,11)-linoleoyl desaturase. FEBS Lett. 462, 249–253. 38. Morris,L.J.,Harris,R.V.,Kelly,W.&James,A.T.(1968)The stereochemistry of desaturation of long-chain fatty acids in Chlorella vulgaris. Biochem. J. 109, 673–678. 39. Rodriguez, S., Camps, F. & Fabrias, G. (2001) Synthesis of gem-dideuterated tetradecanoic acids and their use in investigating the enzymatic transformation of (Z)-11-tetradecenoic acid 5028 D. W. Reed et al. (Eur. J. Biochem. 269) Ó FEBS 2002 into (E,E)-10,12-tetradecadienoic acid. J. Org. Chem. 66, 8052– 8058. 40. Jones, J.P. (1987) The separation of the intermolecular isotope effect for the cytochrome P-450 catalyzed hydroxylation of n-octane into its primary and secondary components. J. Am. Chem. Soc. 109, 2171–2173. 41. Broadwater, J.A., Whittle, E. & Shanklin, J. (2002) Desaturation and Hydroxylation. Residues 148 and 324 of Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of catalytic specificity. J. Biol. Chem. 277, 15613– 15620. Ó FEBS 2002 Calendic acid biosynthesis (Eur. J. Biochem. 269) 5029 . Mechanism of 1,4-dehydrogenation catalyzed by a fatty acid (1,4)-desaturase of Calendula of cinalis Darwin W. Reed 1 , Christopher K. Savile 2 , Xiao. Ontario, Canada The mechanism by which the fatty acid (1,4)-desaturase of Calendula officinalis produces calendic acid from linoleic acid has been probed