Skip to main content
SpringerLink
Log in

Measurement of 2H distribution in natural products by quantitative 2H NMR: An approach to understanding metabolism and enzyme mechanism?

  • Published:
Phytochemistry Reviews Aims and scope Submit manuscript

Abstract

During the biosynthesis of natural products, the intra-molecular distribution of isotopes is introduced as a result of different isotope effects associated with the reactions involved. Due to the sensitivity of certain enzymes to the presence of a heavy isotope, the isotope selection effects related to some transformations can be high, especially for hydrogen. The effect of a series of isotope effects specific to each enzyme-catalysed step are additive during a biosynthetic pathway, leading to fractionation of the isotopes between the starting substrate and the final product. As the individual reactions are acting on different positions in the substrate, the net effect is a non-statistical distribution of isotope within the final product. Quantitative 2H NMR spectroscopy can be used to measure the distribution of 2H at natural abundance in natural products. In the first example, the fermentation of glucose is examined. Glucose can act as a primary carbon source for a wide range of fermentation products, produced by a variety of pathways. In many cases, competing pathways are active simultaneously. The relative fluxes are influenced by both environmental and genetic parameters. Quantitative 2H NMR spectroscopy is being used to obtain mechanistic and regulatory information about isotopic fractionation from glucose during such fermentations. Quantitative 2H NMR spectroscopy can also be used to examine the fractionation in 2H that occurs in long-chain fatty acids during chain elongation and oxygenation. It has been found that the (2H/1H) ratio shows an alternating pattern along the length of the chain and that the residual hydrogen atoms at the sites of desaturation are asymmetrically impoverished. The extent to which the non-statistical distribution of isotopes can be related to the mechanism of enzymes involved in the biosynthetic pathway via kinetic isotopic effects will be discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Aursand M, Mabon F & Martin G (1997) High resolution 1H and 2H NMR spectroscopy of pure and essential fatty acids for plants and animals. Magn. Reson. Chem. 35: S91–S100.

    Google Scholar 

  • Barski O, Gabbay K, Grimshaw C & Bohren K (1995) Mechanism of human aldehyde reductase: Characterization of the active site pocket. Biochemistry 34: 11264–11275.

    PubMed  Google Scholar 

  • Behrouzian B & Buist PH (2003) Bio-organic Chemistry of Plant Lipid Desaturation. Phytochem. Rev.2: this issue.

  • Billault I, Guiet S, Mabon F & Robins R (2001) Natural deuterium distribution in long-chain fatty acids is nonstatistical: A site-specific study by quantitative 2H NMR. ChemBioChem 2: 425–431.

    PubMed  Google Scholar 

  • Billault I, Robins R & Akoka S (2002) Determination of deuterium isotope ratios by quantitative 2H NMR spectroscopy: The ERETICmethod as a generic reference signal. Analyt. Chem. 74: 5902–5906.

    Google Scholar 

  • Bohren K, von Wartburg J-P & Wermuth B (1987) Kinetics of carbonyl reductase from human brain. Biochem. J. 244: 165–171.

    PubMed  Google Scholar 

  • Breitenbach J, Kuntz M, Takaichi S & Sandermann G (1999) Catalytic properties of an expressed and purified higher plant type δ-carotene desaturase from Capsicum annuum. Eur. J. Biochem. 265: 376–383.

    PubMed  Google Scholar 

  • Dennis M (1998) Recent developments in food authentification. Analyst 123: 151R–156R.

    Google Scholar 

  • Duan J-R, Billault I, Mabon F & Robins R (2002) Natural deuterium distribution in fatty acids isolated from peanut seed oil: a site-specific study by quantitative 2H NMR spectroscopy. ChemBioChem 3: 752–759.

    PubMed  Google Scholar 

  • Galliard T & Stumpf P (1966) Fat metabolism in higher plants XXX. Enzymatic synthesis of ricinoleic acid by a microsomal preparation from developing Ricinus communis seeds. J. Biol. Chem. 241: 5806–5812.

    PubMed  Google Scholar 

  • Gonfiantini R, Stichler W & Rozananski K (1995) Standards and intercomparison materials distributed by the International Atomic Energy Agency for stable isotope measurements. In: IAEA, Reference and Intercomparison Materials for Stable Isotopes of Light Elements. Proceedings, consultant Meeting, Vienna, 1–3 December 1993, IAEA-Tecdoc-825 (pp. 13–29), Vienna.

  • Guiet S, Robins RJ, Lees M, Billault I (2003) Quantitative 2HNMR analysis of deuterium distribution in petroselinic acid isolated from parsley seed. Phytochemistry, in press.

  • Harwood J (1997) Plant lipid metabolism. In: Dey P & Harbone J (eds) Plant Biochemistry (pp. 237–272). Academic Press, San Diego.

    Google Scholar 

  • Ikeda Y, Hine D, Okamura-Ikeda K & Tanaka K (1985) Mechanism of action of short-chain, medium-chain acyl-CoA dehydrogenases. J. Biol. Chem. 260: 1326–1337.

    PubMed  Google Scholar 

  • Isaacs N (1987) Kinetic isotope effects. In: Physical Organic Chemistry (pp. 255–281). Longman Group UK Ltd., Harlow, UK.

    Google Scholar 

  • Leadlay P, Albery W & Knowles J (1976) Energetics of triosephosphate isomerase: deuterium isotope effects and the enzymecatalysed reaction. Biochemistry 15: 5617–5620.

    PubMed  Google Scholar 

  • Liemans V, Malaisse-Lagae F, Willem R & Malaisse W (1989) Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates; diastereotopic specificity, isotopic discrimination and intramolecular hydrogen transfer. Biochim. Biophys. Acta 998: 111–117.

    PubMed  Google Scholar 

  • Malaisse W, Malaisse-Lagae F, Liemans V, Ottinger R & Willem R (1990) Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: isotopic discrimination between hydrogen and deuterium. Mol. Cell. Biochem. 93: 153–165.

    PubMed  Google Scholar 

  • Mantel P & Nisbet L (1976) Differentiation of Claviceps purpurea in axenic culture. J. Gen. Microbiol. 93: 321–334.

    PubMed  Google Scholar 

  • Markai S, Marchand P, Mabon F, Baguet E, Billault I & Robins R (2002) Natural Deuterium Distribution in Branched-Chain Medium-Length Fatty Acids is Nonstatistical: a Site-Specific Study by Quantitative 2H NMR Spectroscopy of the Fatty Acids of Capsaicinoids. ChemBioChem 3: 212–218.

    PubMed  Google Scholar 

  • Martin GJ & Martin ML (2003) Climatic significance of isotope ratios. Phytochem. Rev. 2: 179–190.

    Google Scholar 

  • Martin G & Martin M (1981) Deuterium labelling at the natural abundance level as studied by high field quantitative 2H NMR. Tetrahedron Lett. 22: 3525–3528.

    Google Scholar 

  • Martin GJ, Zhang B-L, Naulet, N & Martin M (1986) Deuterium transfer in the bioconversion of glucose to ethanol studied by specific isotope labeling at the natural abundance level. J. Amer. Chem. Soc. 108: 5116–5122.

    Google Scholar 

  • Martin M & Martin G (1991) Deuterium NMR in the study of sitespecific natural isotope fractionation (SNIF-NMR). In: Diehl P, Fluck E, Günther H, Kosfeld R, & Seelig J (eds), Vol. 23 (pp. 1–61) NMR Basic Principles and Progress. Springer, Berlin.

    Google Scholar 

  • Martin M & Martin G (1999) Site-specific isotope effects and origin inference. Analysis 27: 209–213.

    Google Scholar 

  • McInnes A, Walker J & Wright JLC (1983) Regiochemical distribution of deuterium during fatty acid biosynthesis following incorporation of [2-13C, 2-2H3]acetate: a 13C NMR study of exchange, desaturase and enoyl reductase stereospecificities in three algae and a yeast. Tetrahedron 39: 3515–3522.

    Google Scholar 

  • Melander L & Saunders W (1982) Reaction Rates of Isotopic Molecules. John Wiley & Sons, New York.

    Google Scholar 

  • Meyer J & O'Hagan D (1992) Conversion of 3-fluoroacetate by cell-free extracts of Dichapetalum cymosum. Phytochemistry 31: 2699–2701.

    Google Scholar 

  • Monson K & Hayes J (1982) Carbon isotopic fractionation in the biosynthesis of bacterial fatty acids. Ozonolysis of unsaturated fatty acids as a means of determining the intramolecular distribution of carbon isotopes. Geochim. Cosmochim. Acta 46: 139–149.

    Google Scholar 

  • Moreau R & Stumpf P (1981) Recent studies of the enzymic synthesis of ricinoleic acid by developing castor beans. Plant Physiol. 67: 672–676.

    Google Scholar 

  • Morris L (1967) The mechanism of ricinoleic acid biosynthesis in Ricinus communis seeds. Biochem. Biophys. Res. Commun. 29: 311–315.

    PubMed  Google Scholar 

  • Morris L, Hall S & James A (1966) The biosynthesis of ricinoleic acid in Claviceps purpurea. Biochem. J. 100: 29c–30c.

    Google Scholar 

  • Nidetzky B, Klimacek M & Mayer P (2001) Transient-state and steady-state kinetic studies of the mechanism of NADHdependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis. Biochem. 40: 10371–10381.

    Google Scholar 

  • Ohlrogge J & Browse J (1995) Lipid biosynthesis. Plant J. 7: 957–970.

    Google Scholar 

  • Parikh S, Moynihan D, Xiao G & Tong PJ (1999) Roles of Tyrosine 158 and lysine 165 in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis. Biochemistry 38.

  • Pionnier S & Zhang B-L (2002) Application of 2H NMR to the study of natural site-specific hydrogen isotope transfer between substrate, medium and glycerol in glucose fermentation with yeast. Analyt. Biochem. 307: 138–146.

    PubMed  Google Scholar 

  • Pionnier S, Robins R & Zhang B-L (2003) Natural abundance hydrogen isotope affiliation between the reactants and the products in glucose fermentation with yeast. J. Agric. Food Chem. 51: 2076–2082.

    PubMed  Google Scholar 

  • Rosman K & Taylor P (1997) Isotopic composition of the elements 1997. UPAC bulletin, 1–22.

  • Saito K, Kawaguchi A, Nozoe S, Seyama Y & Okuda S (1982) Differential hydrogen exchange during the fatty acid synthetase reaction: deuterium distribution of fatty acid synthesized from [2-2H2]malonyl-CoA. Biochem. Biophys. Res. Commun. 108: 995–1001.

    PubMed  Google Scholar 

  • Saito K, Kawaguchi A, Seyama Y, Yamakawa T & Okuda S (1981) Steric course of deuterium incorporation from [2-2H2]malonyl-CoA into fatty acid synthetases. J. Biochem. 90: 1697–1704.

    PubMed  Google Scholar 

  • Saunders W (1986) Kinetic isotope effects. In: Saunders W (ed) Investigating Rates and Mechanism in Reactions (pp. 565–611). Technical Chemistry, New York.

    Google Scholar 

  • Schmidt H-L, Kexel H, Butzenlechner M, Schwarz S, Gleixner G, Thimet S, Werner RA & Gensler M (1995) Non-statistical isotopic distribution in natural compounds: mirror of their biosynthesis and key for their origin assignment. In: E Wada, T Yoneyama, M Minagawa, T Ando, & BD Fry (eds), (pp. 17–35) Stable Isotope in the Biosphere. Kyoto University Press, Kyoto, Japan.

    Google Scholar 

  • Schmidt H-L & Gleixner G (1998) Carbon isotope effects on key reactions in plant metabolism and 13C-patterns in natural compounds. In: H. Griffiths (ed.), (pp. 13–25) Stable Isotopes. Integration of Biological, Ecological and Geochemical Processes. BIOS Scientific, Oxford.

    Google Scholar 

  • Schmidt H-L, Werner R & Eisenreich W (2003) Systematics of 2H patterns of natural compounds and its importance for the elucidation of biosynthetic pathways. Phytochem. Rev. 2: 61–85.

    Google Scholar 

  • Schmidt H-L, Werner R & Rossmann A (2001) 18O Pattern and biosynthesis of natural products. Phytochemistry 58: 9–32.

    PubMed  Google Scholar 

  • Schroepfer G & Bloch K (1965) The stereospecific conversion of stearic acid to oleic acid. J. Biol. Chem. 240: 54–63.

    PubMed  Google Scholar 

  • Sedgwick B & Cornforth J (1977) The biosynthesis of long chain fatty acids. Stereochemical differentiation in the enzymic incorporation of chiral acetates. Europ. J. Biochem. 75: 465–479.

    PubMed  Google Scholar 

  • Sedgwick B, Cornforth J, French S, Gray R, Kelstrup E & Willadsen P (1977) The biosynthesis of long-chain fatty acids. Incorporation of radioactivity from stereospecifically tritiated malonyl thiol esters and the stereochemistry of the acetyl-CoA carboxylase reaction. Eur. J. Biochem. 75: 481–495.

    PubMed  Google Scholar 

  • Sedgwick B, Morris C & French S (1978) Stereochemical course of dehydration catalysed by the yeast fatty acid synthetase. J. Chem. Soc. Chem. Commun. 193–194.

  • Shanklin J & Cahoon EB (1998) Desaturation and related modifications of fatty acids. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49: 611–641.

    Google Scholar 

  • Tenailleau E, Lancelin P & Akoka S (2002) Authentication of the origin of vanillin using quantitative natural abundance carbon-13 NMR. Paper presented at the STABLE ISOTOPES WEEK, Université de Nantes, France.

    Google Scholar 

  • van Beek T & Subrtova D (1995) Factors involved in the high pressure liquid chromatographic separation of alkenes by means of argentation chromatography on ion exchangers: overview of theory and new practical developments. Phytochem. Anal. 6: 1–19.

    Google Scholar 

  • Werner R & Schmidt H-L (2002) The in vitro nitrogen isotope discrimination among organic plant compounds. Phytochemistry 61: 465–484.

    PubMed  Google Scholar 

  • Zhang B-L, Buddrus, S & Martin M (2000) Site-specific hydrogen isotope fractionation in the biosyntheis of glycerol. Bioorg. Chem. 28: 1–15.

    Google Scholar 

  • Zhang B-L, Billault I, Li X-B, Mabon F, Remaud G & Martin M (2002) Hydrogen isotopic profile in the characterization of sugars. Influence of the metabolic pathway. J. Agric. Food Chem.50: 1574–1580.

    PubMed  Google Scholar 

  • Zhang B-L, Yunianta & Martin M (1995) Site-specific isotope fractionation in the characterization of biochemical mechanisms. J. Biol. Chem. 270: 16023–11629.

    PubMed  Google Scholar 

  • Zheng R & Blanchard J (2000) Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase. Biochemistry 39: 3708–3717.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Isotopic Fractionation in Metabolism Group, LAIEM, CNRS UMR 6006, University of Nantes, BP 99208, 44322, Nantes, France

    Richard J. Robins, Isabelle Billault, Jia-Rong Duan, Sébastien Guiet, Sébastien Pionnier & Ben-Li Zhang

Authors
  1. Richard J. Robins
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Isabelle Billault
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jia-Rong Duan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sébastien Guiet
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sébastien Pionnier
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ben-Li Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Richard J. Robins.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Robins, R.J., Billault, I., Duan, JR. et al. Measurement of 2H distribution in natural products by quantitative 2H NMR: An approach to understanding metabolism and enzyme mechanism?. Phytochemistry Reviews 2, 87–102 (2003). https://doi.org/10.1023/B:PHYT.0000004301.52646.a8

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/B:PHYT.0000004301.52646.a8

Search

Navigation