2. The evolution and structural anatomy of small molecule metabolism pathways in Escherichia coli. Of Pathways and Proteins Stuart Rison and Sarah Teichmann

3. Questions How are homologous proteins (enzymes) distributed in E. coli metabolism? How does this distribution fit with theories of pathway evolution?

4. Pathway evolution Norman Horowitz, 1945: ‘On the evolution of biochemical syntheses’, Proc. Nat. Acc. Sci. 31:153-157. “Retrograde evolution” Roy Jensen, 1976: ‘Enzyme recruitment in evolution of new function’, Ann. Rev. Microbiol 30:409- 425. “Patchwork evolution”

5. Retrograde evolution [ ]

6. Jensen, 1976: Substrate ambiguity ‘Original pool’ of unregulated and enzymatically versatile proteins Enzymes recruited from the pool Ad hoc pathways Gene duplication and specialisation leads to regulated, specific and efficient pathways

7. Patchwork evolution

8. Why E. coli? An extensively studied model organism Complete genome available Most Small Molecule Metabolism pathways well known and empirically characterised A manageable size Good associated databases

9. Strategy Identify all SMM proteins and the pathway(s) in which they belong Detect homologous proteins by structure or sequence Combine these data to analyse homologous protein distribution in SMM

10. Methods E. coli IMPALA HMM  -BLAST (>75aa) += Evolutionary Relationships Pathways Proteins

11. 566 SMM proteins 442 proteins assigned to 1+ families (78%) 124 unassigned proteins 169 PDB-D families31 ‘sequence’ domain families 200 domain families Domain assignments

12. Chemistry and close substrate Chemistry and substrate Glycogen Catabolism malQ malS malZ pgmmalP glgPamyA  -amylase, 3.2.1.1 phosphoglucomutase, 5.4.2.2 amylomaltase, 2.4.1.25  -amylase, 3.2.1.1 glycogen phosphorylase malodextrin phosphorylase malodextrin glucosidase  -glucosyltransferase Phosphoglucomutase  -amylase, C-term Glycosyltransferases Domains Internal duplication Isozymes

13. Duplications Across Pathways 110 out of 200 families occur in more than one pathway Can exhibit conservation of chemistry, shared cofactor or minor substrate similarity 36 families have close conservation of EC number (Chemistry conserved) 74 families conserve 1 or no EC number; 11 are cofactor-binding families (cofactor, minor substrate)

14. Duplications within and across Pathways 710 domains in 200 families  510 domains have arisen by duplication 232 duplications within pathways to 278 duplications across pathways (Assumption: duplication within pathways wherever possible.)

15. Type of conservation

16. Conclusion: Structural Anatomy 710 domains in 442 proteins of the 566 proteins in E. coli SMM pathways 200 families (3.5 members/family) Most sizeable families are distributed in several pathways

17. Conclusion: Recruitment and Conservation Duplications have taken place between and within pathways to roughly the same degree Duplications occur within most longer pathways: –Isozymes, internal duplications and co- factor binding most common –Chemistry common –Conservation of substrate binding with modified chemistry is rare

18. Conclusions: Pathway evolution Data support a “patchwork evolution” model Little evidence of “retrograde evolution”

19. Conclusions: hum… Recruitment, duplication and evolution of enzymes are constantly taking place so we are always observing a dynamic system Likely to be other evolutionary mechanisms and combinations thereof

20. Future Identification and analysis of novel pathway duplication events Focus on order in pathways: –Stepwise analysis –Doublet/triplet analysis Analysis domain combination in SMM

21. Acknowledgements Sarah A. Teichmann, Dept. Biochemistry, University College London Janet M. Thornton, David Lee, Dept. Crystallography, Birkbeck College and Dept. Biochemistry, University College London Monica Riley, Alida Pelegrini-Toole, Marine Biology Laboratory, Woods Hole, USA Cyrus Chothia, Julian Gough, MRC Laboratory of Molecular Biology, Cambridge, UK