Department of Bioorganic Chemistry
Discrimination of MEP and MVA pathways
Natural 13C/12C
Isotope Ratios
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Dr. Stefan Bartram
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The terpenoid pathway allocation via MEP and MVA has been established by
the feeding of 13C labeled glucose and NMR analysis of isolated products.
More recently, Isotope Ratio Mass Spectroscopy (IRMS) at natural abundance
of 13C/12C
(δ13C [‰]-values), has given similar information, without using
labeled precursors which eventually may shift internal equilibria. The
carbon isotopic ratio depends upon enzyme-kinetic isotope-effects, and these
accumulating along the alternative biosynthetic pathways. Terpenoids derived
from MVA have been shown to be more 13C-depleted than those from MEP.
Monoterpenoids, produced by MEP, were reported to have δ13C-values around
–30 ‰, while sesquiterpenoids, produced by MVA, were shown to have
δ13C-values closer to –40 ‰ (Jux, Gleixner & Boland 2001). However, this
dichotomy in the biosynthetic of monoterpenoids and sesquiterpenoids seems
not to be as strict as originally postulated (Rodriguez-Concepcion & Boronat
2002). We focus on the potential cross-talk between the alternative pathways,
based on δ13C-values of terpenoids naturally emitted from plants.
Gas-chromatography combined with combustion, and IR-MS is being used.
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Labeled terpene precursors
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Stefan Garms
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Stable isotopes are the most widely used chemical technique employed in the
elucidation of biosynthetic pathways. In order to discriminate between both
pathways (MEP/DOX and MVA), isotopically labeled (1H or 13C)
1-deoxy-D-xylulose (fig. 2) are synthesized and applied to plants. After
induction plant volatiles are collected and analyzed by GC-MS.
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fig.2: labeled (1H or
13C)
1-deoxy-D-xylulose
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One result of such an experiment is shown in fig. 3 for the sesquiterpene DMNT using deuterated 1-deoxy-D-xylulose and labeled mevalonic acid (in its
lactonic form). Due to the different numbers of deuterium atoms in both
precursors incorporation of them results in different shifts of mass units,
3 for incorporation of 1-deoxy-D-xyluose (left) and 8 for incorporation of
mevalonic acid (right). The fact that an incorporation into DMNT is observed
for both precursors indicates that in some cases a mixture of both pathways
may occur. |
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fig.3: mass spectra of DMNT after incorporation of
[5,5-2H2]-1-deoxy-D-xylulose (left) and [4,4,6,6,6-2H2]-mevalonic acid
(right).
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Literature:
- Jux A., Gleixner G., Boland W. 2001 Classification of terpenoids
according to the methylerythritolphosphate or the mevalonate pathway with
natural
12C/13C isotope ratios: dynamic allocation of resources in induced
plants Angew. Chem. 40(11): 2091-2093
- Rodriguez-Concepcion, M. Boronat, A. 2002 Elucidation of the methylerythritol phosphate pathway for
isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone
achieved through geonomics, Plant Physiology 130(3): 1079-1089.
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