1. The metabolism problem: ingredients of an emerging theory Eörs Szathmáry & Chrisantha Fernando Collegium BudapestEötvös University Budapest

2. Thanks to Günter!

3. Pathways of supersystem evolution boundary template metabolism M BM B B TB T M TM T M B TM B T INFRABIOLOGICAL SYSTEMS

4. General background to the talk

5. The problems of phylogenetic reconstruction (top-down) LUCA was too advanced Reconstructions (e.g. Delaye et al. OLEB in press) cannot reach deep enough The fact that metabolic enzymes are not well conserved does not mean that they were not there! Scaffolds (pre-RNA, primitive metabolic reactions) may have disappeared without leaving a trace behind!!! A more synthetic approach is needed General evolutionary mechanisms must be sought

6. Benner’s hypothetical ribo-organism (1999) Membrane? Cofactors?

7. This raises a lot of questions How could such a metabolic network build up? Did the environment change or not during the process? What was the nature of the non-enzymatic reactions producing (some of) these metabolites? Is an autotrophic, non-enzymatic metabolism feasible? What are the constraints on metabolic evolution in the context of supersystem evolution?

8. Some elementary considerations OL Environment 1Environment 2 Organic synthesis Life Autotrophy impossible Enzymatic pathways are likely to be radically new inventions L O Autotrophy possible Enzymatic pathways may resemble non- enzymatic ones

9. Further complication of supersystem organization The example of the Template/Boundary system: progressive distinction from the environment Metabolites pass freelyMetabolites are hindered evolution

10. Progressive sequestration Initially only templates are kept in They can evolve catalytic properties Carriers and channels can also evolve Membrane permeability can become progressively restrictive Finally, only a very limited sample of molecules can come in Inner and outer environments differentiate Membrane and metabolism coevolve gradually

11. Evolution of metabolism: primitive heterotrophy with pathway innovation ABCDABCD Necessarily heterotrophic protocell A B C D ABCABC A B C D Evolved enzymatic reaction Assume D is the most complex

12. The final stage of innovation A B C D This could be a heterotroph or autotroph (depending on the nature of A)

13. Evolution of metabolism: primitive autotrophy with pathway retention ABCDABCD A B C D abcdabcd a b c D Retroevolution is also likely because of membrane coevolution

14. Reversible versus irreversible: the control of leakage A C Unfavourable: Vulnerable to depletion in A A C BD Favourable: Resistant to depletion in A

15. Two contrasting modes of enzymatic pathway evolution Horowitz (1945) : retroevolution Ancient non-enzymatic pathway: A  B  C  D Progressive depletion of D, then C, then B, then A Selection pressure for enzyme appearance in this order Homologous enzymes will have different mechanisms Jensen (1976) enzyme recruitment (patchwork) One possible mechanism: ambiguity and progressive evolution of specificity Homologous enzymes will have related mechanisms Enzyme recruitment from anywhere (opportunism)

16. What evidence is there for the two mechanisms? New data using the whole armamentarium of bioinformatics It is about the evolution of PROTEIN enzymatic pathways Could be strongly suggestive for RIBOZYME-aided metabolism (the RNA world)

17. The example of biotin metabolism Light and Kraulis (2004) BMC Bioinformatics Homology: strict cutoff in PSI- BLAST

18. Enzymes as edges: the whole E.coli network is analyzed

19. Minimal path length

20. The most promiscuous 20 compounds Frequency: the number of edges where is shows up

21. Homology versus minimal path length With the 20Without the 20

22. Different types of homologous enzyme pairs Mechanistically similar Mechanistically different

23. A statistical analysis Functionally similar Functionally dissimilar

24. Conclusion There is some evidence for retroevolution BUT the dominant mode seems to fit the patchwork mechanism Same mechanisms might worrk for an RNA world!

25. Patchwork and retroevolution can be made compatible A broader notion of retroevolution proposes just the (the frequent) retrograde appearance of consecutive enzymes, not that they are homologous within a pathway Pathways retroevolving in parallel can recruit enzymes in a pacthwork manner

26. Evolution of catalytic proteins or on the origin of enzyme species by means of natural selection Kacser & Beeby (1984) J. Mol. Evol. A precursor cell containing very few multifunctional enzymes with low catalytic activities is shown to lead inevitably to descendants with a large number of differentiated monofunctional enzymes with high turnover numbers. Mutation and natural selection for faster growth are shown to be the only conditions necessary for such a change to have occurred. The division of labour for enzymes!

27. Evolution of connectivity: Pfeiffer et al. (2005) PloS Biology Enzymes are initially specific for the group transferred but not for the substrates Metabolism is based on group transfer reactions between metabolites Without group transfer (D) only unimolecular reactions

28. An emerging group transfer network the frequency, P(k), of metabolites participating in k reactions is given by k -c, where c is a constant coefficient Hubs (127  126 for group 1) emerge as consequence of selection for growth rate

29. An emerging network without group transfer

30. All these ingredients (and more) must be put together Supersystem evolution Alternative environments Progressive sequestration Duplication and divergence of enzymes Selection for cell fitness Network complexification