Nonstatistical 13C Distribution during Carbon Transfer from Glucose to Ethanol during Fermentation Is Determined by the Catabolic Pathway Exploited

Bayle, Kevin; Akoka, Serge; Remaud, Gérald S.; Robins, Richard J.

doi:10.1074/jbc.m114.621441

Cited by 34 publications

(29 citation statements)

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“…However, accurate measurement of position-specific 13 C/ 12 C ratios has until recently been hampered by technical difficulties (see Ref. 19 for an expanded discussion of these), when suitable conditions for isotope ratio monitoring by 13 C NMR (irm- 13 C NMR) spectrometry applied to medium-sized molecules have been established.…”

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confidence: 99%

Non-statistical 13C Fractionation Distinguishes Co-incident and Divergent Steps in the Biosynthesis of the Alkaloids Nicotine and Tropine

Romek

Remaud

Silvestre

et al. 2016

Journal of Biological Chemistry

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During the biosynthesis of natural products, isotopic fractionation occurs due to the selectivity of enzymes for the heavier or lighter isotopomers. As only some of the positions in the molecule are implicated in a given reaction mechanism, positionspecific fractionation occurs, leading to a non-statistical distribution of isotopes. This can be accessed by isotope ratio monitoring 13 C NMR spectrometry. The solanaceous alkaloids S-(؊)-nicotine and hyoscyamine (atropine) are related in having a common intermediate, but downstream enzymatic steps diverge, providing a relevant test case to: (a) elucidate the isotopic affiliation between carbon atoms in the alkaloids and those in the precursors; (b) obtain information about the kinetic isotope effects of as yet undescribed enzymes, thus to make predictions as to their possible mechanism(s). We show that the position-specific 13 C/ 12 C ratios in the different moieties of these compounds can satisfactorily be related to their known precursors and to the known kinetic isotope effects of enzymes involved in their biosynthesis, or to similar reaction mechanisms. Thus, the pathway to the common intermediate, Nmethyl-⌬ 1 -pyrrolinium, is seen to introduce similar isotope distribution patterns in the two alkaloids independent of plant species, whereas the remaining atoms of each target compound, which are of different origins, reflect their specific metabolic ancestry. We further demonstrate that the measured 13 C distribution pattern can be used to deduce aspects of the reaction mechanism of enzymes still to be identified.Members of the plant family Solanaceae produce a range of alkaloids derived from L-ornithine or L-lysine, several of which are exploited for their recreational and/or pharmaceutical properties, despite their toxicity. S-(Ϫ)-Nicotine, the principle alkaloid of Nicotiana tabacum L. and related species has a long history of use for recreational and ethnopharmaceutical applications. Considered a component of the defensive chemical array of the genus Nicotiana (1, 2), nicotine was widely exploited as an insecticide, eventually being phased out as less toxic neonicotinoids became available. Within a closely related group of plants occurs the tropane alkaloids, of which atropine, hyoscyamine, and scopolamine (hyoscine) have a history of use in mystical ceremonies (3). These compounds are now extensively used in modern medicine, atropine and hyoscyamine as mydriatic agents, scopolamine as an anti-emetic for controlling travel sickness.For both these alkaloid types, the biosynthetic pathways have been extensively investigated at the metabolic, enzymatic, and genetic levels (4, 5). Despite superficial structural dissimilarities, the pyrrolidine moiety of nicotine and tropine (of which atropine or hyoscyamine is the tropoyl ester) have a common precursor in L-ornithine, and share four enzymatic steps to the intermediate N-methyl-⌬

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confidence: 99%

Non-statistical 13C Fractionation Distinguishes Co-incident and Divergent Steps in the Biosynthesis of the Alkaloids Nicotine and Tropine

Romek

Remaud

Silvestre

et al. 2016

Journal of Biological Chemistry

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“…Spectra were obtained with a signal-to-noise ratio of ∼650, from which the distribution of 13 C at each carbon position, the reduced molar fraction f i /F i , was calculated from the areas under the peaks. This was then converted to δ 13 C i (‰) values using the δ 13 C g (‰) obtained by irm-MS (Table 1), as previously described (10,19).…”

Section: Resultsmentioning

confidence: 99%

“…Purification including a final crystallization from aqueous acetone yielded pure [>99.5% (wt/wt)] tramadol. Irm-13 C NMR spectra were acquired using 200 mg of pure tramadol from the field collection in slightly aqueous acetone and the δ 13 C i (‰) values computed as described above (10,19).…”

Section: Resultsmentioning

confidence: 99%

“…Likewise, it can provide evidence to distinguish between different pathways to obtain the same product (9,10).…”

Section: Significancementioning

confidence: 99%

“…The curve fitting was carried out with a Lorentzian mathematical model using Perch Software (Perch NMR Software; University of Kuopio, Kuopio, Finland). Data were processed as described previously, and the δ 13 C i (‰) values were calculated from the reduced molar fraction f i /F i and the δ 13 C g (‰) obtained by isotope ratio measurement by mass spectrometry (10,19).…”

Section: H Nmrmentioning

confidence: 99%

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A retro-biosynthetic approach to the prediction of biosynthetic pathways from position-specific isotope analysis as shown for tramadol

Romek

Nun

Remaud

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Tramadol, previously only known as a synthetic analgesic, has now been found in the bark and wood of roots of the African medicinal tree Nauclea latifolia. At present, no direct evidence is available as to the biosynthetic pathway of its unusual skeleton. To provide guidance as to possible biosynthetic precursors, we have adopted a novel approach of retro-biosynthesis based on the position-specific distribution of isotopes in the extracted compound. Relatively recent developments in isotope ratio monitoring by 13 C NMR spectrometry make possible the measurement of the nonstatistical position-specific natural abundance distribution of 13 C (δ 13 C i ) within the molecule with better than 1‰ precision. Very substantial variation in the 13 C positional distribution is found: between δ 13 C i = −11 and −53‰. Distribution is not random and it is argued that the pattern observed can substantially be interpreted in relation to known causes of isotope fractionation in natural products. Thus, a plausible biosynthetic scheme based on sound biosynthetic principals of precursorsubstrate relationships can be proposed. In addition, data obtained from the 18 O/ 16 O ratios in the oxygen atoms of the compound add support to the deductions made from the carbon isotope analysis. This paper shows how the use of 13 C NMR at natural abundance can help with proposing a biosynthetic route to compounds newly found in nature or those difficult to tackle by conventional means.NMR spectrometry | position-specific isotope analysis | retro-biosynthesis | tramadol | Nauclea latifolia

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The new face of isotopic NMR at natural abundance

Jézéquel

Joubert

Giraudeau

et al. 2016

Magnetic Reson in Chemistry

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The most widely used method for isotope analysis at natural abundance is isotope ratio monitoring by Mass Spectrometry (irm-MS) which provides bulk isotopic composition in H, C, N, O or S. However, in the 1980s, the direct access to Site-specific Natural Isotope Fractionation by Nuclear Magnetic Resonance (SNIF-NMR ) was immediately recognized as a powerful technique to authenticate the origin of natural or synthetic products. The initial - and still most popular - application consisted in detecting the chaptalization of wines by irm- H NMR. The approach has been extended to a wide range of methodologies over the last decade, paving the way to a wide range of applications, not only in the field of authentication but also to study metabolism. In particular, the emerging irm- C NMR approach delivers direct access to position-specific C isotope content at natural abundance. After highlighting the application scope of irm-NMR ( H and C), this article describes the major improvements which made possible to reach the required accuracy of 1‰ (0.1%) in irm- C NMR. The last part of the manuscript summarizes the different steps to perform isotope analysis as a function of the sample properties (concentration, peak overlap) and the kind of targeted isotopic information (authentication, affiliation). Copyright © 2016 John Wiley & Sons, Ltd.

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Nonstatistical 13C Distribution during Carbon Transfer from Glucose to Ethanol during Fermentation Is Determined by the Catabolic Pathway Exploited

Cited by 34 publications

References 36 publications

Non-statistical 13C Fractionation Distinguishes Co-incident and Divergent Steps in the Biosynthesis of the Alkaloids Nicotine and Tropine

Non-statistical 13C Fractionation Distinguishes Co-incident and Divergent Steps in the Biosynthesis of the Alkaloids Nicotine and Tropine

A retro-biosynthetic approach to the prediction of biosynthetic pathways from position-specific isotope analysis as shown for tramadol

The new face of isotopic NMR at natural abundance

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