The stereochemistry of desaturations of long-chain fatty acids in <i>Chlorella vulgaris</i>

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

doi:10.1042/bj1090673

Cited by 72 publications

(32 citation statements)

References 8 publications

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“…The second C-H cleavage appears to be fast as might be expected for any of the three pathways shown in Scheme 1. 20,21 In conclusion, we believe that the methodology described in this paper will prove very useful in the study of other desaturases such as the recently discovered soluble, diiron plant ∆9 desaturases for which no mechanistic information is currently available. 18 It is also interesting to note that in very early experiments involving 9-and 10-monotritiated stearates and a bacterial ∆9 desaturase, a kinetic isotope effect at C-9 but not at C-10 was observed.…”

Section: Methodsmentioning

confidence: 93%

“…Two classes of desaturases catalyze this intriguing transformation: soluble plant enzymes containing a carboxylate-bridged, nonheme diiron center 2 and less well-characterized, nonheme iron, membrane-bound catalysts as represented by the ∆9 desaturase found in Saccharomyces cereVisiae 3 and rat liver. 20,21 In conclusion, we believe that the methodology described in this paper will prove very useful in the study of other desaturases such as the recently discovered soluble, diiron plant ∆9 desaturases for which no mechanistic information is currently available. Some of the possible subsequent steps to the olefin are outlined in Scheme 1.…”

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confidence: 93%

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Use of Deuterium Kinetic Isotope Effects To Probe the Cryptoregiochemistry of Δ9 Desaturation

Buist

Behrouzian

1996

J. Am. Chem. Soc.

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The biological syn-dehydrogenation (desaturation) of fatty acids 1 as exemplified by the ∆9 desaturase-mediated transformation of stearoyl CoA (1) to give oleyl CoA (2) represents one of the more virtuosic displays of enzymatic selectivity. Two classes of desaturases catalyze this intriguing transformation: soluble plant enzymes containing a carboxylate-bridged, nonheme diiron center 2 and less well-characterized, nonheme iron, membrane-bound catalysts as represented by the ∆9 desaturase found in Saccharomyces cereVisiae 3 and rat liver. 4 In light of the mounting evidence 5 that desaturases and hydroxylases are structurally related at the protein level, we have adopted the view that desaturations are initiated by a hydrogen abstraction step similar to that proposed for biohydroxylation. Some of the possible subsequent steps to the olefin are outlined in Scheme 1. 6 We have focussed our attention on the ∆9 desaturase of S. cereVisiae and have tentatively placed the putative iron-oxo oxidizing species near C-9 of the substrate since this enzyme system consistently oxygenates 9-thia fatty acid analogues more efficiently than the corresponding 10-thia analogues. 12 In this communication, we report the results of a study in which we further investigate the cryptoregiochemistry of yeast ∆9 desaturation by measuring the deuterium isotope effect for each individual C-H bond cleavage. 13 In order to expedite our isotope effect study, we decided to run direct competition experiments involving methyl 7-thiastearate-9,9-d 2 (3-9,9-d 2 ) vs methyl 7-thiastearate (3) and methyl 7-thiastearate-10,10-d 2 (3-10,10-d 2 ) vs methyl 7-thiastearate (3). Use of methyl stearate-d 2 /d 0 mixtures would have complicated the analysis of the methyl oleate-d 1 /d 0 product due to mass spectral interference by endogenous (d 0 ) oleate. The sulfur atom was placed at position 7 in order to facilitate the synthesis of the deuterated substrates. We have shown previously that methyl 7-thiastearate (3) is converted to the corresponding thiaoleate product (4). 14 3-9,9-d 2 and 3-10,10-d 2 were synthesized in 10% and 8% overall yield, respectively, using well-known procedures as shown in Scheme 2. 15 The two deuterated substrates consisted entirely of dideuterated species (within experimental error) as determined by MS; the 1 H and 13 C NMR spectra were consistent with the location of deuterium label. 3 was available from our previous study. A ca. 1:1 mixture of each deuterated substrate and d 0 material (25 mg) was administered as 5% w/v ethanolic solutions to growing cultures (150 mL) of S. cereVisiae ATCC 12341 as previously described. 12 Each incubation was carried The putative radical intermediate 7 could follow at least three different pathways: one-electron oxidation/H + elimination (pathway a), 8a,b disproportionation (pathway b), 9 or a hydroxyl rebound 10a (SH2) 10b /fast Fe 3+promoted dehydration sequence (pathway c). In addition, the possibility that organoiron intermediates 11 (not shown) are involved in these reactions cannot be...

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Section: Methodsmentioning

confidence: 93%

mentioning

confidence: 93%

Use of Deuterium Kinetic Isotope Effects To Probe the Cryptoregiochemistry of Δ9 Desaturation

Buist

Behrouzian

1996

J. Am. Chem. Soc.

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“…The above proposed stereospecific hydrogen abstraction mechanism accounts for the concerted formation of cis ‐unsaturated bonds, an exquisite power of fatty acid desaturases [65], for which a mechanism has not been previously proposed. Since the microsomal fatty acid desaturases are also known to introduce cis ‐double bonds, they may operate via a similar concerted mechanism.…”

Section: Proposed Involvement Of α‐Tocopherolquinone In Fatty Acid Dementioning

confidence: 94%

A function for the vitamin E metabolite α‐tocopherol quinone as an essential enzyme cofactor for the mitochondrial fatty acid desaturases

Infante¹

1999

FEBS Letters

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A critical analysis of the changes in fatty acid patterns and their metabolism elicited by vitamin E deficiency leads to the proposal that a major role of dietary RRR-K Ktocopherol (K K-TOC) is as an enzymatic precursor of K Ktocopherolquinone (K K-TQ) whose semiquinone radical functions as an essential enzyme cofactor for the fatty acid desaturases of the recently elucidated carnitine-dependent, channeled, mitochondrial desaturation-elongation pathway; a detailed mechanism for its function is proposed. Pathophysiological states produced by vitamin E deficiency and K K-TOC transfer protein defects, such as ataxia, myopathy, retinopathy, and sterility are proposed to develop from the effects of impaired K K-TQdependent desaturases and the resulting deficiency of their polyenoic fatty acid products.z 1999 Federation of European Biochemical Societies.

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“…vulgaris (strain 211/8K) was from the Culture Collection of Algae and Protozoa (Cambridge, UK). 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: Incubation Experimentsmentioning

confidence: 99%

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

Cited by 72 publications

References 8 publications

Use of Deuterium Kinetic Isotope Effects To Probe the Cryptoregiochemistry of Δ9 Desaturation

Use of Deuterium Kinetic Isotope Effects To Probe the Cryptoregiochemistry of Δ9 Desaturation

A function for the vitamin E metabolite α‐tocopherol quinone as an essential enzyme cofactor for the mitochondrial fatty acid desaturases

Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris

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