The stereospecificity of desaturations of long-chain fatty acids in Chlorella vulgaris

Morris, L. J.; Harris, R. V.; Kelly, William T.; James, A. T.

doi:10.1016/0006-291x(67)90064-2

Cited by 24 publications

(15 citation statements)

References 6 publications

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“…2). Thus a synchronous mechanism for the Chlorella Δ 9 desaturase (see Introduction) is ruled out as this would require the operation of isotope effects of similar magnitude for both C–H bond‐breaking steps as apparently had been found previously using an in vivo Chlorella system [16] and a subsequent in vitro desaturase preparation [30]. We believe that the origin of the disagreement between our results and those obtained by previous workers in the case of the Δ 9 desaturase‐mediated reaction can be attributed to their use of erythro‐ and threo ‐9,10‐dideuterated (tritiated) stearoyl substrates − probes which were originally designed to test the stereochemistry of desaturation.…”

Section: Resultsmentioning

confidence: 99%

“…To re‐examine the C. vulgaris Δ 9 desaturase system, we needed to establish the viability of our approach using conditions similar to those of the original experiments [16]. Thus a trial incubation involving the administration of methyl 7‐thiastearate 1 (75 mg·L −1 ) to mid‐logarithmic cells of C. vulgaris (strain 211/8K) was carried out.…”

Section: Resultsmentioning

confidence: 99%

“…This strain of Chlorella is considered identical to that used by Morris et al . [16]. A typical incubation experiment was carried out as follows.…”

Section: Methodsmentioning

confidence: 99%

“…The KIE signatures for both classes of desaturases differ from that apparently obtained by Morris et al . [16] who suggested that primary deuterium isotope effects are expressed at both C‐9 and C‐10 for Δ 9 desaturation as it occurs in the green alga Chlorella vulgaris. This in turn has led to extensive speculation that some desaturases may operate by a synchronous removal of the two vicinal hydrogens [17–20] − a possibility which deserves critical examination.…”

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confidence: 99%

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Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris

Behrouzian

Fauconnot

Daligault

et al. 2001

European Journal of Biochemistry

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The hypothesis that the D 9 desaturase of Chlorella vulgaris might operate by a synchronous mechanism has been tested using a kinetic isotope effect (KIE) approach. Thus the intermolecular primary deuterium KIE on the individual C±H bond cleavage steps involved in D 9 desaturation have been determined by incubating growing cultures of C. vulgaris (strain 211/8K) with mixtures of the appropriate regiospecifically deuterated fatty acid analogues. Our analysis shows that the introduction of a double bond between C-9 and C-10 occurs in two discrete steps as the cleavage of the C9±H bond is very sensitive to isotopic substitution (k H /k d 6.6^0.3) whereas a negligible isotope effect (k H /k d 1.05^0.05) was observed for the C10±H bond-breaking step. Similar results were obtained for linoleic acid biosynthesis (D 12 desaturation). These data clearly rule out a synchronous mechanism for these reactions.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…This strain of Chlorella is considered identical to that used by Morris et al . [16]. A typical incubation experiment was carried out as follows.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris

Behrouzian

Fauconnot

Daligault

et al. 2001

European Journal of Biochemistry

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“…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 Δ 12 ‐desaturation [38].…”

Section: Resultsmentioning

confidence: 99%

Mechanism of 1,4‐dehydrogenation catalyzed by a fatty acid (1,4)‐desaturase of Calendula officinalis

Reed

Savile

Qiu

et al. 2002

European Journal of Biochemistry

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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 produced via initial H-atom abstraction at C11 of a linoleoyl substrate and supports the hypothesis that this transformation represents a regiochemical variation of the more common 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 transformations [1,2]. The prototypical reaction features the introduction 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][7][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)-dehydrogenation 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 hyd...

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Stereochemistry of Δ11-desaturation and inhibitors of Δ10,12-desaturation in the biosynthesis of bombykol, sex pheromone of the female silkworm moth, examined with deuterated precursors

Ando

Ikemoto

Ohno

et al. 1998

Arch. Insect Biochem. Physiol.

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Bombykol [(10E,12Z)‐10,12‐hexadecadien‐1‐ol], the sex pheromone of the silkworm moth (Bombyx mori L.), is biosynthesized from hexadecanoate by three successive steps; i.e., Δ11‐desaturation, Δ10,12‐desaturation, and reduction of the acyl group. 11,12‐Erythro‐ and 11,12‐threo‐[7,8,11,12‐D4]hexadecanoic acid (D4‐16:Acid) were synthesized via the catalytic hydrogenation of the corresponding 7,11‐diene compounds with a Wilkinson's catalyst under deuterium gas and applied to the pheromone gland of B. Mori. GC‐MS analysis of their conversion into bombykol and (Z)‐11‐hexadecen‐1‐ol, another component in the pheromone gland, revealed that the Δ11‐desaturation proceeded in syn‐elimination. A previous experiment using [16,16,16‐D3]16:Acid has shown that some cyclopropene fatty acids and the amides inhibit the desaturation steps of bombykol biosynthesis. In order to define the structure‐activity relationships, [13,13,14,14, 15,15,16,16,16‐D9](Z)‐11‐hexadecenoic acid (D9‐Z11‐16:Acid) was synthesized from D10‐1‐butanol; and the effect of cyclopropene compounds on the incorporation of D9‐Z11‐16:Acid into bombykol was examined utilizing a selected ion monitoring technique of GC‐MS. The result confirmed that Δ10,12‐desaturation was strongly inhibited by 11,12‐methylenehexadec‐11‐enoic acid and the amide, and moderately by 13,14‐methylene derivatives, while 7,8‐ and 9,10‐methylene derivatives did not inhibit this desaturation. Arch. Insect Biochem. Physiol. 37:8–16, 1998. © 1998 Wiley‐Liss, Inc.

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The stereospecificity of desaturations of long-chain fatty acids in Chlorella vulgaris

Cited by 24 publications

References 6 publications

Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris

Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris

Mechanism of 1,4‐dehydrogenation catalyzed by a fatty acid (1,4)‐desaturase of Calendula officinalis

Stereochemistry of Δ11-desaturation and inhibitors of Δ10,12-desaturation in the biosynthesis of bombykol, sex pheromone of the female silkworm moth, examined with deuterated precursors

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