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Daniel E. Almonacid and Patricia C. Babbitt

Classification of Mechanistically Diverse Enzyme Superfamilies According to Similarities in Reaction Mechanism. Daniel E. Almonacid and Patricia C. Babbitt. 18 th July 2008. Overview. Introduction Motivation E.C. Classification Enzyme Catalysis Databases

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Daniel E. Almonacid and Patricia C. Babbitt

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  1. Classification of Mechanistically Diverse Enzyme Superfamilies According to Similarities in Reaction Mechanism Daniel E. Almonacid and Patricia C. Babbitt 18th July 2008

  2. Overview • Introduction • Motivation • E.C. Classification • Enzyme Catalysis Databases • Structure-Function Linkage Database • Methods • Enolase Superfamily • Computing Similarity of Mechanisms and Overall Reactions • Results • Overall vs Mechanism Similarity • Complete Linkage Clustering • Applications • Conclusions

  3. Motivation • Enzymes catalyse almost all the reactions in the metabolism of all organisms. • Knowledge about the evolution of structure-function relationships in enzymesallows prediction of function for newly obtained sequences and structures, and to direct enzyme engineering efforts.

  4. E.C. Classification Enzyme Commission (EC) Nomenclature, 1992, Academic Press, 6th Ed.

  5. Enzyme Catalysis Databases Pegg, S. C.-H., et al. Biochemistry, 2006, 45, 2545 Holliday, G. L., et al. Nucleic Acids Res., 2007, 35, D515

  6. SFLD (http://sfld.rbvi.ucsf.edu/)

  7. Enolase Superfamily

  8. Enolase Superfamily

  9. Enolase Superfamily

  10. Enolase Superfamily

  11. Enolase Superfamily

  12. Enolase Superfamily

  13. Enolase Superfamily

  14. Dataset Labeling E1 GD1 MR1 . . .

  15. Computing Mechanism Similarity chloromuconate cycloisomerase (MC6) dipeptide epimerase (MC2)

  16. Similarity between Reaction Steps chloromuconate cycloisomerase (MC6) • Bond order changes: • C-O C=O • C=C  C-C • C-C  C=C • Bonds formed: • None • Bonds cleaved: • C-O Step 3 dipeptide epimerase (MC2) • Bond order changes: • C-O C=O • C=C  C-C • Bonds formed: • C-H • Bonds cleaved: • Base-H Step 2 Step similarity (Tanimoto coeff) = intersection / union = 2/(4+4-2) = 0.3333

  17. Global Alignment of Reaction Sequences a) Similarity Matrix b) Needleman-Wunsch Maximum-Match Matrix

  18. Similarity between Reaction Mechanisms chloromuconate cycloisomerase (MC6) dipeptide epimerase (MC2) Step 1 Step 2 Step 3 Step 1 Step 2 1.0 0.3333 Alignment score, Axy, of 1.3333 normalised similarity, Sxy = Axy Axx + Ayy - Axy Sxy = 1.3333 = 0.3636 3 + 2 – 1.3333 NM O’Boyle, et al., J. Mol. Biol., 2007, 368, 1484.

  19. Overall vs Mechanistic Similarity • A total of 190 pairs are compared. • Size of the spheres is proportional to the number of data points in that position. • Significance levels are shown in red.

  20. Similarity between Overall Reactions chloromuconate cycloisomerase (MC6) dipeptide epimerase (MC2)

  21. Similarity between Overall Reactions chloromuconate cycloisomerase (MC6) • Bond order changes: • None • Bonds formed: • C-Cl • Bonds cleaved: • C-O Overall dipeptide epimerase (MC2) • Bond order changes: • None • Bonds formed: • C-H • Bonds cleaved: • C-H Overall Overall similarity (Tanimoto coeff) = intersection / union = 0/(4+4-0) = 0

  22. Overall vs Mechanistic Similarity • A total of 190 pairs are compared. • Size of the spheres is proportional to the number of data points in that position. • Significance levels are shown in red.

  23. Complete Linkage Clustering of Mechanisms Common partial reaction: chloromuconate cycloisomerase (SFLD10) dipeptide epimerase (SFLD12)

  24. Same Subgroup, Different Mechanism Common partial reaction: chloromuconate cycloisomerase (SFLD10) chloromuconate cycloisomerase dipeptide epimerase dipeptide epimerase (SFLD12)

  25. Different Subgroup, Same Mechanism Common partial reaction: o-succinyl- benzoate synthase Enolase D-tartrate dehydratase

  26. Different Subgroup, Same Mechanism D-tartrate dehydratase (MR1) enolase (E1) o-succinylbenzoate synthase (MC1)

  27. Preliminary Anecdotal Observation Common partial reaction: muconate cycloisomerase o-succinyl- benzoate synthase dipeptide epimerase

  28. Preliminary Anecdotal Observation Target: o-succinylbenzoate synthase (MC1) dipeptide epimerase (MC2) muconate cycloisomerase (MC7) Schmidt, et al., Biochemistry, 2003, 42, 8387.

  29. Conclusions • Compared to the traditional approach of classifying enzymes according to overall reaction similarity (such as that of the Enzyme Commission), the method based on step similarity is better able to capture elements of functional conservation. • The relationship between sequence/structure and function is yet more complicated than previously envisaged. • We expect our study to be useful for guiding functional annotation of new homologues of enzyme superfamilies, and to help guide engineering of enzyme functions by identifying enzyme templates capable of catalyzing the key mechanistic step of a transformation

  30. Acknowledgements Margy Glasner Sunil Ojha Shoshana Brown Patricia Babbitt Gemma Holliday Janet Thornton Noel O’Boyle John Mitchell $$$ NIH NSF ISCB

  31. Questions? • daniel.almonacid@ucsf.edu • Structure-Function Linkage Database • http://sfld.rbvi.ucsf.edu/

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