Evolution & organisation of metabolic pathways

1. Evolution & organisation of metabolic pathways Bas Kooijman Dept of Theoretical Biology Vrije Universiteit, Amsterdam http://www.bio.vu.nl/thb/deb/ Dynamic Energy Budget theory for metabolic organisation embryo adult juvenile Amsterdam, 2004/03/31 the dynamic structure of life

2. Central Metabolism source polymers monomers waste/source

3. Modules of central metabolism • Pentose Phosphate (PP) cycle • glucose-6-P ribulose-6-P, • NADP NADPH • Glycolysis • glucose-6-P pyruvate • ADP + P ATP • TriCarboxcyl Acid (TCA) cycle • pyruvate CO2 • NADP NADPH • Respiratory chain • NADPH + O2 NADP + H2O • ADP + P ATP

4. Evolution of central metabolism in prokaryotes (= bacteria) 3.8 Ga 2.7 Ga i = inverse ACS = acetyl-CoA Synthase pathway PP = Pentose Phosphate cycle TCA = TriCarboxylic Acid cycle Kooijman, Hengeveld 2003 The symbiontic nature of metabolic evolution Acta Biotheoretica (to appear) RC = Respiratory Chain Gly = Glycolysis

5. Prokaryotic metabolic evolution • Heterotrophy: • pentose phosph cycle • glycolysis • respiration chain • Phototrophy: • el. transport chain • PS I & PS II • Calvin cycle • Chemolithotrophy • acetyl-CoA pathway • inverse TCA cycle • inverse glycolysis

6. Early ATP generation • FeS + S0  FeS2 • ADP + Pi ATP • ATPase • hydrogenase • S-reductase FeS2 FeS 2H+ H2 S0 H2S ADP ATP 2e- Pi S0 H2S 2H2O 2H+ 2OH- Madigan et al 1997

7. Substrate processing Fractions of SU ·· unbound A· SU-A complex ·B SU-B complex AB SU-A,B complex Synthesizing Units: generalized enzymes process arriving fluxes of substrate reversed flux is small mixtures of processing schemes are possible Kooijman, 2001

8. Biomass: reserve(s) + structure(s) • Reserve(s), structure(s): generalized compounds, • mixtures of proteins, lipids, carbohydrates: fixed composition • Reserve(s) do complicate model & implications & testing • Reasons to delineate reserve, distinct from structure • metabolic memory • biomass composition depends on growth rate • explanation of • respiration patterns (freshly laid eggs don’t respire) • method of indirect calorimetry • fluxes are linear sums of assimilation, dissipation and growth • inter-species body size scaling relationships • fate of metabolites (e.g. conversion into energy vs buiding blocks)

9. Reserve vs structure • Reserve does not mean: “set apart for later use” • compounds in reserve can have active functions • Life span of compounds in • reserve: limited due to turnover of reserve • all reserve compounds have the same mean life span • structure: controlled by somatic maintenance • structure compounds can differ in mean life span • Important difference between reserve and structure: • no maintenance costs for reserve • Empirical evidence: • freshly laid eggs consist of reserve and do not respire

10. Homeostasis Homeostasis: constant body composition in varying environments Strong homeostasis generalized compounds applies to reserve(s) and structure(s) separately Weak homeostasis: ratio reserve/structure becomes and remains constant if food or substrate is constant (while the individual is growing) applies to juvenile and adult stages, not to embryos Implication: stoichiometric constraints on growth

11. Methanotrophs CO2 Macro-chemical reaction at fixed growth rate reserve NH3 • DEB decomposition into • assimilation (substrate  reserve) • catabolic & anabolic aspect • maintenance (reserve  products) • growth (reserve  structure) • catabolic & anabolic aspect • yield coefficients vary with growth • reserve, structure differ in composition • composition of biomass varies with growth CH4 O2 Kooijman, Andersen & Kooi 2004

12. Anammox Macro-chemical reaction at r = 0.0014 h-1 • DEB decomposition into • assimilation (substrate  reserve) • catabolic & anabolic aspect • maintenance (reserve  products) • growth (reserve  structure) • catabolic & anabolic aspect • yield coefficients vary with growth • reserve, structure differ in composition • composition of biomass varies with growth • rm = 0.003 h-1; kE = 0.0127 h-1; kM = 0.0008 h-1 • ySE = 8.8; yVE = 0.8 • nHE = 2; nOE = 0.46; nNE = 0.25 • nHV = 2; nOV = 0.51; nNV = 0.125 Brandt, 2002

13. Nitrogen cycle Brocadia anammoxidans some cyanobacteria, Azotobacter, Azospirillum, Azorhizobium, Klebsiella, Rhizobium,some others Nitrosomonas Some crucial conversions depend on few species Nitrobacter many CHON= biomass

14. Syntrophy Coupling hydrogen & methane production energy generation aspect at aerobic/anaerobic interface ethanol dihydrogen acetate bicarbonate methane dihydrogen Total: methane hydrates >300 m deep, < 8C linked with nutrient supply

15. Product Formation According to Dynamic Energy Budget theory: Product formation rate = wA. Assimilation rate + wM. Maintenance rate + wG . Growth rate For pyruvate: wG<0 ethanol pyruvate, mg/l pyruvate glycerol, ethanol, g/l glycerol Applies to all products, heat & non-limiting substrates Indirect calorimetry (Lavoisier, 1780): heat = wO JO + wC JC + wN JN No reserve: 2-dim basis for product formation throughput rate, h-1 Glucose-limited growth of Saccharomyces Data from Schatzmann, 1975

16. Symbiosis substrate product

17. Symbiosis substrate substrate

18. Steps in symbiogenesis Internalization Free-living, clustering Free-living, homogeneous Reserves merge Structures merge

19. Chemostat Steady States Free living Products complementary Endosymbiosis Exchange on conc-basis Free living Products substitutable biomass density Exchange on flux-basis Structures merged Reserves merged Host uses 2 substrates throughput rate symbiont host

20. Symbiogenesis • symbioses: fundamental organization of life based on syntrophy • ranges from weak to strong interactions; basis of biodiversity • symbiogenesis: evolution of eukaryotes (mitochondria, plastids) • DEB model is closed under symbiogenesis: • it is possible to model symbiogenesis of two initially independently • living populations that follow the DEB rules by incremental changes • of parameter values such that a single population emerges that • again follows the DEB rules • essential property for models that apply to all organisms • Kooijman, Auger, Poggiale, Kooi 2003 • Quantitative steps in symbiogenesis and the evolution of homeostasis • Biological Reviews78: 435 - 463

21. Symbiogenesis 1.5-2 Ga 1.2 Ga

22. Eukaryote metabolic evolution First eukaryotes: heterotrophs by symbiogenesis compartmental cellular organisation Acquisition of phototrophy frequently did not result in loss of heterotrophy Acquisition of membrane transport between internalization of mitochondria and plastids No phagocytosis in fungi & plants; loss? pinocytosis in animals = phagocytosis in e.g. amoeba? Direct link between phagocytosis and membrane transport?

23. Membrane traffic The golgi apparatus serves as a central clearing house and channel between the endo- and exoplasmic domains 1 ER-Golgi shuttle 2 secretory shuttle between Golgi and plasma membrane 2’ crinophagic diversion 3 Golgi-lysosome shuttle 3’ alternative route from Golgi to lyosomes via the plasma membrane and an endosome 4 endocytic shuttle between the plasma membrane and an endosome 4’ alternative endocytic pathway bypassing an endosome 5 plasma membrane retrieval 6 endosome-lysosome pathway 7 autophagic segregation From: Duve, C. de 1984 A guided tour of the living cell, Sci. Am. Lib., New York

24. Clathrin unknown in prokaryotes

25. Chloroplast dynamics Coordinated movement of chloroplasts through cells

26. Composed by Bas Kooijman (brown algae) Phaeophyceae Granuloreticulata forams Xenophyophora Basidiomycota Xanthophyceae Raphidophyceae Retaria Ascomycota fungi Chrysophyceae Synurophyceae Glomeromycota Actinopoda Eustigmatophyceae Zygomycota Labyrinthulomycota Dictyochophyceae Microsporidia Bicosoecia Pedinellophyceae Opisthokonts Chytridiomycota Pelagophyceae Bigyromonada Plasmodiophoromycota Bacillariophyceae (diatoms) Cercozoa Chlorarachnida Pseudofungi Chromista Cercomonada animals Bolidophyceae Opalinata animals Choanozoa Prymnesiophyceae loss phagoc. Metamonada Cryptophyceae Apusozoa gap junctions tissues (nervous) mitochondria Sporozoa Alveo- lates (plants) Cormophyta bicentriolar mainly chitin EF1 insertion Percolozoa primary Myxomycota Excavates Dinozoa chloroplast Protostelida secondary (green algae) Chlorophyceae Ciliophora Bikont DHFR-TS gene fusion Amoebozoa Euglenozoa chloroplast Archamoeba Plantae tertiary mainly celllose membr. dyn unikont cortical alveoli (red algae) Rhodophyceae chloroplast Rhizopoda chloroplasts triple roots Loukozoa photo Bacteria Bacteria symbionts Glaucophyceae Sizes of blobs do not reflect number of species Survey of organisms

27. Cells, individuals, colonies vague boundaries • plasmodesmata connect cytoplasm; cells form a symplast: plants • pits and large pores connect cytoplasm: fungi, rhodophytes • multinucleated cells occur; individuals can be unicellular: • fungi, Eumycetozoa, Myxozoa, ciliates, Xenophyophores, Actinophryids, Biomyxa, diplomonads, • Gymnosphaerida, haplosporids, Microsporidia, nephridiophagids, Nucleariidae, plasmodiophorids, • Pseudospora, Xanthophyta (e.g. Vaucheria), most classes of Chlorophyta (Chlorophyceae, Ulvophyceae, • Charophyceae (in mature cells) and all Cladophoryceae, Bryopsidophyceae and Dasycladophyceae)) • cells inside cells: Paramyxea • uni- and multicellular stages: multicellular spores in unicellular myxozoa, gametes • individuals can remain connected after vegetative propagation: plants, corals, bryozoans • individuals in colonies can strongly interact • and specialize for particular tasks: • syphonophorans, insects, mole rats Kooijman, Hengeveld 2003 The symbiontic nature of metabolic evolution Acta Biotheoretica (to appear) rotifer Conochilus hippocrepis Heterocephalus glaber

28. (Endo)symbiosis Frequent association between photo- and heterotroph photo  hetero: carbohydrates (energy supply) photo  hetero: nutrients (frequently NH3 or NO3-) most (perhaps all) plants have myccorrhizas, the symbiosis combines photolithotrophy and organochemotrophy Also frequent: association between phototroph and N2-fixer where N2-fixer plays role of heterotroph Symbiosis: living together in interaction (basic form of life) Mutualism: “benefit” for both partners symbioses need not be mutualistic “benefit” frequently difficult to judge and anthropocentric Syntrophy: one lives of products of another (e.g. faeces) can be bilateral; frequent basis of symbiosis

29. Chlorochromatium (Chlorobibacteria, Sphingobacteria) (= Chlorochromatium) From: Margulis, L & Schwartz, K.V. 1998 Five kingdoms.Freeman, NY

30. (Endo)symbiosis Paramecium bursaria ciliate with green algae Cladonia diversa ascomycete with green algae Ophrydium versatile ciliate with green algae Peltigera ascomycete with green algae

31. (Endo)symbiosis Chlorophyte symbionts visible through microscope Grazed by reindeer in winter Rangifer tarandus Lichen Cladonia portentosa

32. Mitochondria TriCarboxylic Acid cycle (= Krebs cycle) Enzymes pass metabolites directly to other enzymes enzymes catalizing transformations 5 & 7: bound to inner membrane (and FAD/FADH2) Net transformation: Acetyl-CoA + 3 NAD+ + FAD + GDP 3- + Pi2- + 2 H2O = 2 CO2 + 3 NADH + FADH2 + GTP 4- + 2 H+ + HS-CoA Dual function of intermediary metabolites building blocks  energy substrate all eukaryotes once possessed mitochondria, most still do enzymes are located in metabolon; channeling of metabolites Transformations: 1 Oxaloacetate + Acetyl CoA + H2O = Citrate + HSCoA 2 Citrate = cis-Aconitrate + H2O 3 cis-Aconitrate + H2O = Isocitrate 4 Isocitrate + NAD+ = α-Ketoglutarate + CO2+ NADH + H+ 5 α-Ketoglutarate + NAD+ + HSCoA = Succinyl CoA + CO2 + NADH + H+ 6 Succinyl CoA + GDP 3- + Pi 2- + H+ = Succinate + GTP 4- + HSCoA 7 Succinate + FAD = Fumarate + FADH2 8 Fumarate + H2O = Malate 9 Malate + NAD+ = Oxaloacetate + NADH + H+

33. Pathways & allocation structure structure maintenance maintenance reserve reserve structure Mixture of products & intermediary metabolites that is allocated to maintenance (or growth) has constant composition reserve maintenance Kooijman & Segel, 2004

34. Numerical matching for n=4 0 4 1 3 2 2 3 Unbound fraction 4 Product flux 1 Spec growth rate • = 0.73, 0.67, 0.001, 0.27 handshaking • = 0.67, 0.91, 0.96, 0.97 binding prob k = 0.12, 0.19, 0.54, 0.19 dissociation nSE = 0.032,0.032,0.032,0.032 # in reserve nSV = 0.045,0.045,0.045,0.045 # in structure yEV= 1.2 res/struct kE= 0.4 res turnover jEM = 0.02 maint flux n0E = 0.05 sub in res 0 Rejected flux 1 2 3 Spec growth rate

35. Matching pathway  whole cell No exact match possible between production of products and intermediary metabolites by pathway and requirements by the cell But very close approximation is possible by tuning abundance parameters and/or binding and handshaking parameters Good approximation requires all four tuning parameters per node growth-dependent reserve abundance plays a key role in tuning Kooijman, S. A. L. M. and Segel, L. A. (2004) How growth affects the fate of cellular substrates. Bull. Math. Biol. (to appear)