1. The Evolution of Metabolism Chrisantha Fernando School of Computer Science Birmingham University 17th November 2005. Systems Biology Group Meeting Chrisantha Fernando School of Computer Science Birmingham University 17th November 2005. Systems Biology Group Meeting

2. Pre-Enzymatic Metabolic Evolution Part 1.

3. Pathways of supersystem evolution boundary template metabolism M BM B B TB T M TM T M B TM B T Pre-enzymatic and post-enzymatic stages can be distinguished.

4. Background

5. All living systems today have metabolism.  An organism without metabolism would be one that did not synthesize any of its constituents, but obtained them all preformed from the environment or from its parent(s).  Heterotrophic theories of the origin of life (Oparin, Haldane, Lancet, Eigen, Kauffman, Farmer, Fox, Szostak) assume such ‘organisms’ were possible.  An organism without metabolism would be one that did not synthesize any of its constituents, but obtained them all preformed from the environment or from its parent(s).  Heterotrophic theories of the origin of life (Oparin, Haldane, Lancet, Eigen, Kauffman, Farmer, Fox, Szostak) assume such ‘organisms’ were possible.

6. The Problem of 1 o Heterotrophy.  Initial bolus of complex organics from space.  Chemical energy used to form organism depletes this bolus.  Low gross primary production of complex organics (because no autotrophs).  Therefore any 1 o heterotroph exists in an ecological transient, and can be saved only by the evolution of an autotroph.  In the long term, [metabolic entities] are rate limiting to non-metabolic entities.  Initial bolus of complex organics from space.  Chemical energy used to form organism depletes this bolus.  Low gross primary production of complex organics (because no autotrophs).  Therefore any 1 o heterotroph exists in an ecological transient, and can be saved only by the evolution of an autotroph.  In the long term, [metabolic entities] are rate limiting to non-metabolic entities.

7. All known cellular life has an autocatalytic metabolism.  Remove all metabolites, leaving water and informational macromolecules in place + ATP. The network cannot be re-created from the food materials alone.  All cells possess a distributive autocatalytic network, that cannot be seeded from outside, because some of its seed components cannot be taken up (or synthesized) from the medium.  Remove all metabolites, leaving water and informational macromolecules in place + ATP. The network cannot be re-created from the food materials alone.  All cells possess a distributive autocatalytic network, that cannot be seeded from outside, because some of its seed components cannot be taken up (or synthesized) from the medium.

8. The Chemoton (T. Ganti 1971)

9. Benefits of autocatalytic metabolism.  During hard times, key metabolites cannot escape, whereas the non-autocatalytic entity could loose its metabolites by reactions running in reverse.  Contemporary metabolic networks are endogenously autocatalytic.  The unit of chemical evolution is the autocatalytic cycle, (existing within a recycling system).  During hard times, key metabolites cannot escape, whereas the non-autocatalytic entity could loose its metabolites by reactions running in reverse.  Contemporary metabolic networks are endogenously autocatalytic.  The unit of chemical evolution is the autocatalytic cycle, (existing within a recycling system).

10. How likely is it for autocatalytic cycles to arise and persist?  Imagine an experiment, C,H,N,O,P,S, heterogeneous environments and flux keeps system away from equilibrium.  Under what circumstances will the system settle down into a boring point attractor (tar) and when will it produce life?  G.A.M. King. Selection of rate coefficients and concentrations of reagents are needed to make anything but the smallest cycle to persist. [The problem of side-reactions/specificity].  Imagine an experiment, C,H,N,O,P,S, heterogeneous environments and flux keeps system away from equilibrium.  Under what circumstances will the system settle down into a boring point attractor (tar) and when will it produce life?  G.A.M. King. Selection of rate coefficients and concentrations of reagents are needed to make anything but the smallest cycle to persist. [The problem of side-reactions/specificity].

11. Pre-enzymatic symbiosis of autocatalytic particles.  If symbiosis can reduce the rate of decay of the symbiont (i.e. increase specificity), then even if the growth rate of the symbiont is lower than its components, the coupled cycle has a selective advantage.  But a successful symbiosis is not easy, (“The Good Symbiont”, ECAL 2005).  If symbiosis can reduce the rate of decay of the symbiont (i.e. increase specificity), then even if the growth rate of the symbiont is lower than its components, the coupled cycle has a selective advantage.  But a successful symbiosis is not easy, (“The Good Symbiont”, ECAL 2005).

13. Modeling Chemical Evolution.  A model of a heterogenious, interestingly structured, platonic, chemical space which can be explored.  Ensure conservation of mass and energy.  Allow niche selection in chemical space.  Allow physical niche selection (i.e. selection of abiotic catalysts, and selection of diffusion limiting lipid membranes).  The system must be capable of discovery of scaffolding/channeling systems that reduce side- reactions.  A model of a heterogenious, interestingly structured, platonic, chemical space which can be explored.  Ensure conservation of mass and energy.  Allow niche selection in chemical space.  Allow physical niche selection (i.e. selection of abiotic catalysts, and selection of diffusion limiting lipid membranes).  The system must be capable of discovery of scaffolding/channeling systems that reduce side- reactions.

15. Possible approaches so far…  Gil Benko.  My DPhil Chapter 2.  The problem is to develop a valid model of chemistry in which chemical evolution can be explored.  Then to expose this model to appropriate environments.  Efforts underway in Germany to Ix. Formose cycle metabolism evolution.  Gil Benko.  My DPhil Chapter 2.  The problem is to develop a valid model of chemistry in which chemical evolution can be explored.  Then to expose this model to appropriate environments.  Efforts underway in Germany to Ix. Formose cycle metabolism evolution.

16. Post-Enzymatic Metabolic Evolution Part 2.

17. General Assumptions  Assume an underlying (non/minimal-enzymatic) metabolism in a protocell capable of synthesizing ribozymes.  Since there is no relationship between the catalytic power of a given RNA and the protein for which it encodes, there is no clear path from the RNA to the protein world. Therefore protein cladistics reach a historical limit.  Evolution of metabolism is by genetic assimilation of underlying chemical pathways.  Several evolutionary motifs have been proposed.  Assume an underlying (non/minimal-enzymatic) metabolism in a protocell capable of synthesizing ribozymes.  Since there is no relationship between the catalytic power of a given RNA and the protein for which it encodes, there is no clear path from the RNA to the protein world. Therefore protein cladistics reach a historical limit.  Evolution of metabolism is by genetic assimilation of underlying chemical pathways.  Several evolutionary motifs have been proposed.

18. i) Horowitz’s Retro-extension ABCDABCD Necessarily heterotrophic protocell A B C D ABCABC A B C D Evolved enzymatic reaction Assume D is the most complex

19. A B C D The final stage of innovation

20. Horowitz’s assumptions and their consequences.  D is indeed available at an early stage.  C,B, and A are available in excess in the environment.  This is only likely where autotrophs produce them.  Therefore, retroevolution may be important when a heterotroph co-evolves closely with an autotroph.  Retroevolution is also likely due to membrane co- evolution, i.e. where D can no longer enter, and so must be synthesized.  D is indeed available at an early stage.  C,B, and A are available in excess in the environment.  This is only likely where autotrophs produce them.  Therefore, retroevolution may be important when a heterotroph co-evolves closely with an autotroph.  Retroevolution is also likely due to membrane co- evolution, i.e. where D can no longer enter, and so must be synthesized.

21. Wachtershauser’s Operations.

22. Loss of pathways.  A  B  C  D : A  B  Using various combinations of the above primitives, predictions can be made about evolutionary trajectories leading to extant metabolic systems.  However the possible trajectories are underdetermined, so additional assumptions are required.  A  B  C  D : A  B  Using various combinations of the above primitives, predictions can be made about evolutionary trajectories leading to extant metabolic systems.  However the possible trajectories are underdetermined, so additional assumptions are required.

23. Assume Evolutionary Opportunism  Melendez-Hevia et al.

24. Is there an evolutionary trace of the actual trajectory? 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) 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)

25. Light and Kraulis (2004)  Homologous enzyme pairs abound at the minimum path length of one, (i.e. the product of one is the substrate of the other).  But does not corroborate retro-extension because,  Only small homology between mpl 2 and 3 pairs.  Most enzyme pairs with mpl 1 have similar EC numbers, hence are functionally related.  Retro-extension may still have been important in the RNA world.  Homologous enzyme pairs abound at the minimum path length of one, (i.e. the product of one is the substrate of the other).  But does not corroborate retro-extension because,  Only small homology between mpl 2 and 3 pairs.  Most enzyme pairs with mpl 1 have similar EC numbers, hence are functionally related.  Retro-extension may still have been important in the RNA world.

26. With 20 promis.Without 20 promis. Function SimilarFunction Dissimilar.

27. But patchwork and retro-extension are not mutually exclusive.  A broader notion of retroevolution proposes just the (frequent) retrograde appearance of consecutive enzymes, not that they are homologous within a pathway  Pathways retroevolving in parallel can recruit enzymes in a patchwork manner  A broader notion of retroevolution proposes just the (frequent) retrograde appearance of consecutive enzymes, not that they are homologous within a pathway  Pathways retroevolving in parallel can recruit enzymes in a patchwork manner

28. Why Scale-Free? - Preferential attachment (Light, Kraulis, Elofsson 2005).  Older enzymes are more highly connected (duplic and diverged enzyme may preserve past reagents).  HGT enzymes are more highly connected (may aid retention? - Preferential attachment (Light, Kraulis, Elofsson 2005).  Older enzymes are more highly connected (duplic and diverged enzyme may preserve past reagents).  HGT enzymes are more highly connected (may aid retention?

29. The origin of enzyme species by 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.  Duplication and divergence and natural selection for faster growth are shown to be the only conditions necessary for such a change to have occurred.  Assumes that increasing the copy number of one enzyme gene decreases numbers of other enzymes.  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.  Duplication and divergence and natural selection for faster growth are shown to be the only conditions necessary for such a change to have occurred.  Assumes that increasing the copy number of one enzyme gene decreases numbers of other enzymes.

30. From K&B  The thermodynamic constraints within which cells operate do not define the particular kinetic organization that we observe.  A clever student of biochemistry could invent a variety of metabolic maps and associated enzymes which differ substantially from those now.  Pathways existed prior to the arrival of enzymes. Their presence allows the kinetic realization of a particular subset of all thermodynamically possible steps. Another set of enzymes would produce another map.  The thermodynamic constraints within which cells operate do not define the particular kinetic organization that we observe.  A clever student of biochemistry could invent a variety of metabolic maps and associated enzymes which differ substantially from those now.  Pathways existed prior to the arrival of enzymes. Their presence allows the kinetic realization of a particular subset of all thermodynamically possible steps. Another set of enzymes would produce another map.

31. 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  Supersystem evolution  Alternative environments  Progressive sequestration  Duplication and divergence of enzymes  Selection for cell fitness  Network complexification

32. What should we do?  Make a model of the underlying platonic metabolic pathways, coupled with a model of enzyme evolution, and cell level fitness subject to reasonable environmental conditions.  How in principle could such systems co-evolve?  E.g. Pfeiffer et al showed co-evolution of increasing specific group transfer metabolite co- enzymes with their specific enzymes, in a group- transfer network, selected for growth rate.  Make a model of the underlying platonic metabolic pathways, coupled with a model of enzyme evolution, and cell level fitness subject to reasonable environmental conditions.  How in principle could such systems co-evolve?  E.g. Pfeiffer et al showed co-evolution of increasing specific group transfer metabolite co- enzymes with their specific enzymes, in a group- transfer network, selected for growth rate.

33. Biasing Assumptions.  1. They assumed that initially ALL enzymes were present capable of catalysing all possible metabolic group transfers. A more reasonable possibility is that no genetically encoded enzymes are present (only non- encoded catalysts), but that there is an inherent rate of underlying group transfer reactions. In the model there is no underlying metabolic thermodynamics or kinetics.  2. Enzyme kinetics were capable of being arbitrarily uniformly mutated, with free energies of forward and backward reactions being easy to alter arbitrarily by mutation. Is this reasonable? Could an enzyme really be so flexible in its functional response to mutation?  3. They assume that enzyme concentrations cannot be influenced by end products, that enzymes cannot be inhibited or activated by normal substrates. Not so bad for a first attempt.  4. They assume that enzymes specificity is for acceptor and donor groups, but that there is no evolution of enzyme specificity for the catalysed group itself, rather this remains constant throughout. Is this reasonable?  5. Increasing the copy number of one enzyme gene did not decrease the concentrations of other enzymes.  6. They assume that the environment over all evolutionary history consists of 0000000 and 1111111, therefore not properly testing the scenarios would have allowed evolution by retro-extension.  1. They assumed that initially ALL enzymes were present capable of catalysing all possible metabolic group transfers. A more reasonable possibility is that no genetically encoded enzymes are present (only non- encoded catalysts), but that there is an inherent rate of underlying group transfer reactions. In the model there is no underlying metabolic thermodynamics or kinetics.  2. Enzyme kinetics were capable of being arbitrarily uniformly mutated, with free energies of forward and backward reactions being easy to alter arbitrarily by mutation. Is this reasonable? Could an enzyme really be so flexible in its functional response to mutation?  3. They assume that enzyme concentrations cannot be influenced by end products, that enzymes cannot be inhibited or activated by normal substrates. Not so bad for a first attempt.  4. They assume that enzymes specificity is for acceptor and donor groups, but that there is no evolution of enzyme specificity for the catalysed group itself, rather this remains constant throughout. Is this reasonable?  5. Increasing the copy number of one enzyme gene did not decrease the concentrations of other enzymes.  6. They assume that the environment over all evolutionary history consists of 0000000 and 1111111, therefore not properly testing the scenarios would have allowed evolution by retro-extension.