Application of KIE and thia approaches in the mechanistic study of a plant stearoyl-ACP Δ9 desaturase

Behrouzian, Behnaz; Buist, Peter H.; Shanklin, John

doi:10.1039/b008972i

Cited by 33 publications

(37 citation statements)

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“…Interestingly, the deuterium profile measured by 2 H NMR in chiral oriented media of methyl linoleate isolated from a plant (sunflower) shows similar ( 2 H/ 1 H) 9 and ( 2 H/ 1 H) 13 ratios, whereas the ( 2 H/ 1 H) 10 ratio was higher than the ( 2 H/ 1 H) 12 ratio (24). These measurements are in full agreement with the results found for the soluble plant ⌬ 9 -desaturase, where no primary KIE at position 9 has been observed (44).…”

Section: Discussionsupporting

confidence: 82%

Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

Baillif

Robins

Feunteun

et al. 2009

Journal of Biological Chemistry

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The origin of hydrogen atoms during fatty acid biosynthesis in Fusarium lateritium has been quantified by isotope tracking close to natural abundance. Methyl linoleate was isolated from F. lateritium grown in natural abundance medium or in medium slightly enriched with labeled water, glucose, or acetate, and the 2 H incorporation was determined by quantitative 2 H-{ 1 H} NMR in isotropic and chiral oriented solvents. Thus, the individual ( 2 H/ 1 H) i ratio at each pro-R and pro-S hydrogen position of the CH 2 groups along the chain can be analyzed. These values allow the isotope redistribution coefficients (a ij ) that characterize the specific source of each hydrogen atom to be related to the nonexchangeable hydrogen atoms in glucose and to the medium water. In turn, these can be related to the stereoselectivity that operates during the introduction or removal of hydrogens along the fatty acid chain. First, at even CH 2 the pro-S hydrogen comes only from water by protonation, whereas the pro-R hydrogen is introduced partly via acetate but principally from water. Second, the nonexchangeable hydrogens of glucose (positions H-6,6 and H-1) are shown to be introduced to the odd CH 2 via the NAD(P)H pool used by both reductases involved in the elongation steps of the fatty acid chain. Third, it is proved that hydrogens removed at sites 9,10 and 12,13 during desaturation by ⌬ 9 -and ⌬ 12 -desaturases are pro-R, and that during these desaturation steps ␣-secondary kinetic isotope effects occur at the 9 and 12 positions and not at the 10 and 13 positions.Fatty acids are ubiquitous natural products involved in many key biological processes, including acting as components of membranes, as lipophilic modifiers, as fuel stores, and as precursors of intracellular messengers (1-3). Their biosynthesis, which is strictly conserved throughout higher organisms, can be separated into two parts as follows: the formation of the basic C16 unit, palmitoylCoA (C16:1), by the fatty-acid synthase complex (FAS) 2 and the subsequent modification of this chain by a range of elongases, desaturases, conjugases, hydroxylases, and epoxidases. Many of these steps involve stereoselective enzymatic reactions during which hydrogen atoms are inserted or eliminated.We have previously shown that this process leads to a distribution of 2 H in long chain fatty acids that is nonstatistical (4 -7). Thus, isotopic fractionation is seen to be introduced. Two features general to all organisms so far examined can be noted. First, the methylenic groups at even positions tend to be richer than those at odd positions. Second, one of the ethylenic groups present at the positions of desaturation is consistently impoverished relative to the methylenic sites, whereas the other is not.Thus, each hydrogen atom in the final product will have a ( 2 H/ 1 H) ratio that is representative of its initial origin, its passage through the FAS elongation steps, and its potential participation in post-elongation modification of the chain. To date, it has proved possible to exp...

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Section: Discussionsupporting

confidence: 82%

Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

Baillif

Robins

Feunteun

et al. 2009

Journal of Biological Chemistry

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“…The ratio of 9-to 10-sulfoxidized products (1.6 : 1) obtained in this work compares favorably with the values obtained from both direct competition (2.1 : 1 [6]) and noncompetitive incubation of thiastearates (1.7 [5]). Good correlation between sulfoxidation ratios derived from experiments with nonfluorinated and w-fluorinated thiastearates have also been obtained for a soluble, plant D9-desaturase system [9] [15].…”

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confidence: 69%

ω‐Fluoro Thiafatty Acids: New Mechanistic Probes of Desaturase‐Mediated Reactions

Hodgson

Lao

Dawson

et al. 2003

Helvetica Chimica Acta

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Dedicated to Duilio Arigoni on the occasion of his 75th birthday A series of 18-fluoro thiastearates were prepared and incubated with a yeast D9-desaturating system. The relative efficiency of desaturase-mediated sulfoxidation was monitored via 19 F-NMR analysis of the sulfoxide products, and a strong preference for oxo transfer to the S-atom occupying the 9-position was confirmed. The oxidation profile obtained in this manner matched that of analogous experiments with non-fluorinated substrates. These results form the basis of a versatile 19 F-NMR-based method for mapping the position of the putative diiron oxidant relative to substrate, and has potential application to the study of membrane-bound desaturases in vitro.1. Introduction. ± Fatty acid desaturases are an important superfamily of enzymes found in a wide variety of aerobic organisms [1]. The first desaturation reaction to be studied in detail was the conversion of stearoyl CoA to oleyl CoA as it occurs in baker×s yeast (Scheme 1, a) [2]. Interest in this particular enzyme system has risen recently with the discovery that enhanced stearoyl CoA D9 desaturase (SCD) activity in mammals occurs in a number of disorders, including obesity, diabetes, and other metabolic diseases [3]. Thus, the development of mechanism-based SCD inhibitors would have potential therapeutic significance. A major stumbling block to progress in this area is that stearoyl CoA D9 desaturase is membrane-bound and difficult to isolate in purified form. However, some important mechanistic information has been obtained on the structurally related yeast enzyme [4], which can be conveniently studied in vivo by an appropriate experimental design. A KIE study showed that the H-atom at C(9) of substrate is removed first in an isotopically sensitive step, followed by rapid cleavage of the CÀH bond at C(10) [5]. This result correlates well with the observation that desaturase-mediated oxo transfer occurs most efficiently when a S-atom occupies the 9-position of a thiastearoyl substrate (Scheme 1, b) [6]; the enantioselectivity of sulfoxidation matches the stereochemistry (syn, pro-R) of the corresponding parent reaction [7] [8]. Taken together, these results strongly suggest that the putative diiron oxidant involved in yeast D9 desaturase mediated oxidation is asymmetrically located between C(9) and C(10) of substrate.The success of the thia-analogue approach in providing critically important information on desaturase-active-site topology prompted us to consider modifications to the probe that would obviate the need to isolate milligram amounts of product for

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“…5 The thioester products were reductively cleaved with NaBH 4 to give the corresponding terminal alcohols, which were then extracted with dichloromethane (Scheme 2). The solvent was evaporated under N 2 and the entire extract was redissolved in freshly purified CDCl 3 and examined by 19 F NMR.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, we have discovered that this enzyme is capable of sulfoxidizing 9-and 10-thia-substrate analogues, although with markedly different efficiencies (Scheme 1). 5 In order to evaluate fully the regioselectivity of this process, a more complete analysis of all enzymatic products was required. Here, we show how this goal was accomplished through the…”

Section: Introductionmentioning

confidence: 99%

Use of ¹⁹F NMR spectroscopy to probe enzymatic oxidation of fluorine‐tagged sulfides

Behrouzian

Hodgson

Savile

et al. 2002

Magnetic Reson in Chemistry

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A novel19 F NMR-based method for monitoring the enzymatic oxidation of thia fatty acid analogues is presented. Our approach is based on the observation that methyl !-monofluorinated 9-thia-and 10-thiaoctadecanoates and their S-oxide and S-dioxide derivatives are easily distinguishable via their 1 H-decoupled 19 F spectra. These long-range substituent effects were used to probe the regio-and chemoselectivity of stearoyl ACP (acyl carrier protein) 1 9 desaturase-mediated sulfoxidation. The results clearly demonstrate that mono-oxygenation of a 10-thia analogue ACP 1 9 desaturase was more efficient than that of a 9-thia substrate. A product previously undetected by TLC was observed for the first time in the product mixture obtained from 18-fluoro-9-thiaoctadecanoyl-ACP.

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Application of KIE and thia approaches in the mechanistic study of a plant stearoyl-ACP Δ9 desaturase

Cited by 33 publications

References 16 publications

Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

ω‐Fluoro Thiafatty Acids: New Mechanistic Probes of Desaturase‐Mediated Reactions

Use of ¹⁹F NMR spectroscopy to probe enzymatic oxidation of fluorine‐tagged sulfides

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Application of KIE and thia approaches in the mechanistic study of a plant stearoyl-ACP Δ9 desaturase

Cited by 33 publications

References 16 publications

Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

ω‐Fluoro Thiafatty Acids: New Mechanistic Probes of Desaturase‐Mediated Reactions

Use of 19F NMR spectroscopy to probe enzymatic oxidation of fluorine‐tagged sulfides

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Use of ¹⁹F NMR spectroscopy to probe enzymatic oxidation of fluorine‐tagged sulfides