1. Enzyme Evolution John Mitchell, February 2010

2. Theories of Enzyme Evolution

3. Overview (1) Divergent retrograde evolution, recruiting adjacent enzymes in pathway and constrained by binding similar molecules as substrates or products.

4. Overview (2) Divergent patchwork evolution, recruiting enzymes catalysing similar chemical reactions, typically from other pathways, constrained by supporting similar catalytic chemistry.

5. Overview (3) Convergent evolution, reinventing similar chemistry in a different evolutionary family.

6. Retrograde Evolution (Horowitz,1945) Pathways evolve backwards: the end product of the newly evolved reaction is the substrate of the existing one.

7. Picture adapted from Betts & Russell, 2009

9. Successive reactions in the pathway would therefore be catalysed by homologous enzymes Picture adapted from Betts & Russell, 2009

10. Patchwork Evolution (Jensen,1976) Recruitment of enzymes for new reactions was based on similarity of reactions catalysed and possibly on substrate ambiguity. It did not necessarily require the sequential and backwardly evolving progression of steps.

11. Patchwork recruitment: Recruit an enzyme with a chemically similar catalytic function from a quite different pathway.

12. Picture from Betts & Russell, 2009

15. The Importance of Moonlighting A moonlighting enzyme has a second job.

16. The Importance of Moonlighting Patchwork recruitment is most likely to occur when the original enzyme already has some low level of activity for catalysing a different reaction.

17. The Importance of Moonlighting This allows the enzyme to be recruited to carry out the new function.

18. Main reaction Minor side reaction

19. A starting point for evolving a new catalytic function! Main reaction Minor side reaction

20. How Might Divergent Evolution Occur? At the level of the gene, the most obvious idea is via gene duplication with one copy being free to mutate away from its original function

21. Picture adapted from Todd, Orengo & Thornton, 1999

23. Original functionNew function

24. Picture adapted from Todd, Orengo & Thornton, 1999 Original functionNew function This way, the original function is maintained and a new one evolved.

25. How Might Divergent Evolution Occur? There are other possible routes to diverged functions (Orengo, Thornton, Todd & others)

26. Picture adapted from Todd, Orengo & Thornton, 1999

27. Models for Divergent Enzyme Evolution Two main models of divergent enzyme evolution discussed by Gerlt and Babbitt: Chemistry is conserved, substrate specificity changes. Substrate binding is conserved, chemistry changes. Evidence for both models in different cases, but conserved chemistry is likely to be more common.

28. Models for Divergent Enzyme Evolution Two main models of divergent enzyme evolution discussed by Gerlt and Babbitt: Chemistry is conserved, substrate specificity changes. If true, implies that chemical reactions are harder to evolve than is substrate binding. Fits well with patchwork recruitment model.

29. Enzyme Nomenclature and Classification EC Classification Class Subclass Sub-subclass Serial number

30. Phylogeny of Enzymes (Caetano-Annolés) Taking advantage of the genomic data now available, Caetano-Annolés and group attempted to build a phylogeny of enzymes based on the occurrence of their folds in sequenced genomes.

31. Phylogeny of Enzymes (Caetano-Annolés) In principle, this could “age” enzymes – the more universal the older.

32. Picture from Caetano-Annolés et al. (2007)

33. The diverse mix of “ages” within metabolic networks seems to support the patchwork model. Picture from Kim et al. (2006)

34. Using similar ideas of universality of superfamilies, tried to reconstruct proteome of LUCA. Interesting, but speculative?

35. Both Divergent & Convergent Evolution are Important Divergent evolution leads to one fold performing a plurality of functions. Convergent evolution leads to a plurality of folds performing the same function

36. Takes advantage of folds being both structural and evolutionary units of protein structure.

38. Divergence

39. Convergence

40. Those figures are based on available structures. As more become available, we will find more functions for existing folds, and more folds with existing functions. So these are underestimates! Convergent Divergent

41. Caveat: Our working definition of “Convergent Evolution” is dependent on the EC classification, which is not a perfect gold standard.

42. MACiE Mechanism, Annotation and Classification in Enzymes. http://www.ebi.ac.uk/thornton-srv/databases/MACiE/ The MACiE Database G.L. Holliday et al., Nucl. Acids Res., 35, D515-D520 (2007) Gemma Holliday, Daniel Almonacid, Noel O’Boyle, Janet Thornton, Peter Murray-Rust, Gail Bartlett, James Torrance, John Mitchell

44. Enzyme Nomenclature and Classification EC Classification Class Subclass Sub-subclass Serial number

45. The EC Classification  Deals with overall reaction, not mechanism  Reaction direction arbitrary  Cofactors and active site residues ignored  Doesn’t deal with structural and sequence information  However, it was never intended to do so

46. A New Representation of Enzyme Reactions?  Should be complementary to, but distinct from, the EC system  Should take into account:  Reaction Mechanism  Structure  Sequence  Active Site residues  Cofactors  Need a database of enzyme mechanisms

47. MACiE Mechanism, Annotation and Classification in Enzymes. http://www.ebi.ac.uk/thornton-srv/databases/MACiE/ MACiE Database

50. Difficulties of Hierarchical Classification Very similar mechanisms can end up in different first level classes. In the case of phosphoinositide-specific phospholipases C, this is due to a slow final hydrolysis step occurring in one of the two enzymes.

51. Classifying Related Enzymes: Phosphoinositide-specific Phospholipases C Eukaryotic (rat) Cell Signalling Multidomain Catalytic TIM Barrel EC 3.1.4.11 Hydrolase Final hydrolysis step Prefers bisphosphate Acid-base mechanism Calcium dependent Prokaryotic (B. cereus) Virulence factor Single domain Catalytic TIM Barrel EC 4.6.1.13 Lyase No/slow final hydrolysis Disfavours bisphosphate Acid-base mechanism Not calcium dependent Evolutionarily related

52. Similar reactions end up far apart EC 3.1.4.11 EC 4.6.1.13

53. Difficulties of Hierarchical Classification Different mechanisms can occur with exactly the same EC number. MACiE has six beta-lactamases, all with different mechanisms but the same overall reaction.

55. MACiE Mechanisms are Sourced from the Literature

56. Coverage of MACiE Representative – based on a non-homologous dataset, and chosen to represent each available EC sub-subclass.

57. EC Coverage of MACiE Representative – based on a non-homologous dataset, and chosen to represent each available EC sub-subclass. Structures exist for: 6 EC 1.-.-.- 57 EC 1.2.-.- 194 EC 1.2.3.- 1547 EC 1.2.3.4 MACiE covers: 6 EC 1.-.-.- 54 EC 1.2.-.- 165 EC 1.2.3.- 249 EC 1.2.3.4

58. Convergent Evolution of Enzyme Function D.E. Almonacid et al., PLoS Computational Biology, accepted N.M. O’Boyle et al., J. Molec. Biol., 368, 1484-1499 (2007)

59. We use a combination of bioinformatics & chemoinformatics to identify similarities between enzyme-catalysed reaction mechanisms

60. Similarity of Overall Reactions: Compare Bond Changes

61. Similarity of Mechanisms: Compare Steps

62. Just like sequence alignment! We can measure their similarity … Similarity of Mechanisms: Compare Steps

63. Carrying out an analysis of pairwise similarity of reactions in MACiE...

64. Find only a few similar pairs

65. Identify convergent evolution

66. Check MACiE for duplicates!

67. Mechanistic similarity is only weakly related to proximity in the EC classification

68. EC in common 0 -.-.-.- 1 c.-.-.- 2 c.s.-.- 3 c.s.ss.-

69. Similarity of Analogous Reactions We take all possible pairs of analogous enzyme reactions from MACiE 2.3.9 Analogous means that they carry out similar functions (EC 1.2.3.- conserved)...... and that the enzymes are not homologous We find 95 analogous pairs (convergent evolution).

73. 43 out of 95 pairs that are analogous according to EC have no significant reaction or mechanistic similarity Shared EC sub-subclass and Bond Change based reaction similarity are quite different criteria.

74. Has implications for using EC as a gold standard of similarity in studies of evolution. Shared EC sub-subclass and Bond Change based reaction similarity are quite different criteria.

76. One third of analogous pairs with significantly similar overall reactions have significantly similar mechanisms.

77. Most often, similar overall reactions that evolve convergently will have quite different mechanisms.

79. For analogous pairs, we find that mechanistic similarity is less than overall similarity (almost always); these lie in the lower triangle.

80. Conclusions for Analogous Enzymes Conservation of EC sub-subclass does not imply quantitative reaction similarity. One third of analogous pairs with significantly similar overall reactions have significantly similar mechanisms. Mechanistic similarity is less than overall similarity (unlike homologues).

81. Theories of Enzyme Evolution

82. Overall Conclusions Both convergent and divergent evolution are important. Patchwork evolution is quite common. Retrograde evolution does sometimes occur (adjacent reactions in pathways have similar enzymes more often than by chance), but is not strongly prevalent. Consistent with the above, conservation of chemistry is more important than conservation of substrate binding.

83. ACKNOWLEDGEMENTS Cambridge Overseas Trust