Unfinished business from April 4! Unfinished business from April 4! Metabolomics, spring 06 Hans Bohnert ERML 196 [email protected]265-5475 333-5574 http://www.life.uiuc.edu/bohnert/ Metabolite profiling = a static picture, a snapshot! Does it matter?* nie AR et al. (2005) Flux an important, but neglected, component of functional genomics. Curr. Opin. Plant Biology 8, 174. l DA (2005) Enzymes, metabolites and fluxes. J. Exptl Botany 56, 267 nd two case studies. class April 6
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Unfinished business from April 4! Metabolomics, spring 06 Hans Bohnert ERML 196 [email protected] 265-5475 333-5574
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Unfinished business from April 4!Unfinished business from April 4!Metabolomics, spring 06
Can we infer flux from changes in intermediates? think allosteric effects of metabolites measuring regulated steps in a pathway is intermediates [conc](consider the Mark Stitt lecture)
Pathways branch (label lost)Different pathway(s) provide(s) intermediate (label diluted by unknown)Tracer addition may change the equilibrium of the systemPlants: where, and how, to introduce the tracerPool size – dilution of labelIs end-product transported – loss of labelDo we know the pathway, or assume we know, and are we right
Need certainty about pathway structures – (MapMan, TAIR, KEGG) – do we?Need certainty about pathway structures – (MapMan, TAIR, KEGG) – do we?
Measuring end-product – stable, transported or metabolized (e.g., disappear in cell wall; does CO2 production and glycolysis)
Branched pathways – do we know
Linear relationship between product level and time (growth!)
Experimental material – entire plant, organ (or part of organ), tissue slice, cells, organelles
How “big” is the flux, the pathway – can we actually measure it? NMR (stable isot.), GC-MS, LC-MS - sensitivity and accuracy
Positional information of tracer substrate modification may be important
Long-term feeding expt, or pulse labeling, or pulse/chase expts
Schwender et al. (2004) Rubisco without the Calvin cycle improves the carbon efficiency of developing green seeds. Nature 432, 779. (on web as: Shachar-Hill-Nature-2004)
Figure 1a
Figure 1b
Figure 1 Metabolic transformation of sugars into fatty acids. a, Conversion of hexose phosphate to pentose phosphate through the non-oxidative steps of the pentose phosphate pathway and the subsequent formation of PGA by Rubisco bypasses the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase while recycling half of the CO2 released by PDH. PGA is then further processed to pyruvate, acetyl-CoA and fatty acids.
b, Part of a expanded to indicate carbon skeletons and to define relationships between
V PDH (flux through PDH complex); V X (additional CO2 production by the OPPP, the TCA, and so on); V Rub (refixation by Rubisco). Metabolites: Ac-CoA, acetyl coenzyme-A; DHAP, dihydroxyacetone-3-phosphate; E4P, erythrose-4-phosphate; Fru-6P, fructose-6-phosphate; GAP, glyceraldehydes-3-phosphate; Glc-6P, glucose-6-phosphate; PGA, 3-phosphoglyceric acid; Pyr, pyruvate; R-5P, ribose-5-phosphate; Ru-1,5-P2, ribulose-1,5-bisphosphate; Ru-5P, ribulose-5-phosphate; S-7P, sedoheptulose-7-phosphate; Xu-5P, xylulose-5-phosphate. Enzymes: Aldo, fructose bisphosphate aldolase; Eno, 2-phosphoglycerate enolase; Xepi, xylulose-5-phosphate epimerase; FAS, fatty-acid synthase, PGM, phosphoglyceromutase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, phosphoglucose isomerase; Riso, ribose-5-phosphate isomerase; PDH, pyruvate dehydrogenase; PFK, phosphofructokinase; PK, pyruvate kinase, PGK, phosphoglycerate kinase; PRK, phosphoribulokinase; TA, transaldolase; TK, transketolase; TPI, triose phosphate isomerase.
Conclusions Rubisco operates as part of a previously undescribed metabolic routebetween carbohydrate and oil (Fig. 1a). Three stages:(1) conversion of hexose phosphates to ribulose-1,5-bisphosphate by the non-oxidative reactions of the OPPP together with phosphoribulokinase.
(2) conversion of ribulose-1,5-bisphosphate and CO2 (most produced by PDH3) to PGA by Rubisco
(3) metabolism of PGA to pyruvate and then to fatty acids (Fig. 1a).
The net carbon stoichiometry of this conversion: 5 hexose phosphate > 6 pentose phosphate > 12 acetyl-CoA + 6 CO2
The conversion of the same amount of hexose phosphates by glycolysis:5 hexose phosphate >10 Acetyl-CoA + 10 CO2
Roessner-Tunali et al. (2004) Kinetics of labeling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes. Plant J. 39, 668.
Where does the label go?
• Primary metabolism
• potato tubers
• wild type and transgenics
• EI GC-MS
• U-13C/14C glucose feeding
• pathway verification
Possible reaction ratesto measure
What is U-13C or U-14C glucose?
Wt
INV-2-30
SP-29
Amounts over time (up to 12h)
(P < 0.05)
bold - transgenic difference to wild type
important – watch differences in rates of synthesis (Δf = >100)