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

2. Central Metabolism polymers monomers waste/source source

3. 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 CO 2 NADP NADPH Respiratory chain NADPH + O 2 NADP + H 2 O ADP + P ATP Modules of central metabolism

4. Evolution of central metabolism i = inverse ACS = acetyl-CoA Synthase pathway PP = Pentose Phosphate cycle TCA = TriCarboxylic Acid cycle RC = Respiratory Chain Gly = Glycolysis Kooijman, Hengeveld 2003 The symbiontic nature of metabolic evolution Acta Biotheoretica (to appear) in prokaryotes (= bacteria) 3.8 Ga2.7 Ga

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

6. Early ATP generation FeS 2 FeS H2H2 2H + H2SH2S S0S0 S0S0 H2SH2S 2OH - 2H + ADP ATP PiPi 2e - 2H 2 O FeS + S 0  FeS 2 ADP + P i  ATP ATPase hydrogenase S-reductase Madigan et al 1997

7. Synthesizing Units: generalized enzymes process arriving fluxes of substrate reversed flux is small mixtures of processing schemes are possible Substrate processing Fractions of SU  ·· unbound  A· SU-A complex  ·B SU-B complex  AB SU-A,B complex 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. 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 Methanotrophs Kooijman, Andersen & Kooi 2004 Macro-chemical reaction at fixed growth rate CO 2 NH 3 CH 4 O2O2 reserve

12. 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 r m = 0.003 h -1 ; k E = 0.0127 h -1 ; k M = 0.0008 h -1 y SE = 8.8; y VE = 0.8 n HE = 2; n OE = 0.46; n NE = 0.25 n HV = 2; n OV = 0.51; n NV = 0.125 Anammox Brandt, 2002 Macro-chemical reaction at r = 0.0014 h -1

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

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

15. Product Formation throughput rate, h -1 glycerol, ethanol, g/l pyruvate, mg/l glycerol ethanol pyruvate Glucose-limited growth of Saccharomyces Data from Schatzmann, 1975 According to Dynamic Energy Budget theory: Product formation rate = w A. Assimilation rate + w M. Maintenance rate + w G. Growth rate For pyruvate: w G <0 Applies to all products, heat & non-limiting substrates Indirect calorimetry (Lavoisier, 1780): heat = w O J O + w C J C + w N J N No reserve: 2-dim basis for product formation

16. Symbiosis product substrate

17. Symbiosis substrate

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

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

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 Reviews 78: 435 - 463

21. Symbiogenesis 1.5-2 Ga1.2 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 From: Duve, C. de 1984 A guided tour of the living cell, Sci. Am. Lib., New York 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

24. Clathrin unknown in prokaryotes

25. Chloroplast dynamics Coordinated movement of chloroplasts through cells

26. Survey of organisms mitochondria secondary chloroplast primary chloroplast tertiary chloroplast Sizes of blobs do not reflect number of species Bacteria Opisthokonts Chromista Amoebozoa Alveo- lates Plantae Excavates Retaria Cercozoa fungi animals forams cortical alveoli Bikont DHFR-TS gene fusion chloroplasts membr. dyn unikont loss phagoc. gap junctions tissues (nervous) bicentriolar mainly chitin EF1  insertion triple roots mainly celllose photo symbionts

27. Cells, individuals, colonies 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 vague boundaries 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 NH 3 or NO 3 - ) most (perhaps all) plants have myccorrhizas, the symbiosis combines photolithotrophy and organochemotrophy Also frequent: association between phototroph and N 2 -fixer where N 2 -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) From: Margulis, L & Schwartz, K.V. 1998 Five kingdoms.Freeman, NY (= Chlorochromatium)

30. (Endo)symbiosis Paramecium bursaria ciliate with green algae Ophrydium versatile ciliate with green algae

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

32. Mitochondria Transformations: 1 Oxaloacetate + Acetyl CoA + H 2 O = Citrate + HSCoA 2 Citrate = cis-Aconitrate + H 2 O 3 cis-Aconitrate + H 2 O = Isocitrate 4 Isocitrate + NAD + = α-Ketoglutarate + CO 2 + NADH + H + 5 α-Ketoglutarate + NAD + + HSCoA = Succinyl CoA + CO 2 + NADH + H + 6 Succinyl CoA + GDP 3- + P i 2- + H + = Succinate + GTP 4- + HSCoA 7 Succinate + FAD = Fumarate + FADH 2 8 Fumarate + H 2 O = Malate 9 Malate + NAD + = Oxaloacetate + NADH + H + TriCarboxylic Acid cycle (= Krebs cycle) Enzymes pass metabolites directly to other enzymes enzymes catalizing transformations 5 & 7: bound to inner membrane (and FAD/FADH 2 ) Net transformation: Acetyl-CoA + 3 NAD + + FAD + GDP 3- + P i 2- + 2 H 2 O = 2 CO 2 + 3 NADH + FADH 2 + 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

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

34. Numerical matching for n=4 Product flux Rejected flux Unbound fraction  = 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 n SE = 0.032,0.032,0.032,0.032 # in reserve n SV = 0.045,0.045,0.045,0.045 # in structure y EV = 1.2 res/struct k E = 0.4 res turnover j EM = 0.02 maint flux n 0E = 0.05 sub in res 0 0 1 1 1 2 2 2 3 3 3 4 4 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)