1. 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
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 enzymes allows prediction of function for newly obtained sequences and structures, and to direct enzyme engineering efforts.

4. Enzyme Commission (EC) Nomenclature, 1992, Academic Press, 6th Ed.
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)
Bonds formed:
None
Bonds cleaved:
C-O
Bond order changes:
C-O C=O
C=C  C-C
C-C  C=C
Step 3
dipeptide epimerase (MC2)
Bonds formed:
C-H
Bonds cleaved:
Base-H
Bond order changes:
C-O C=O
C=C  C-C
Step 2
Step similarity (Tanimoto coeff) = intersection / union = 2/(4+4-2) =

17. Global Alignment of Reaction Sequences
a) Similarity Matrix
MC2.stg01
MC2.stg02
MC6.stg01
0.0000
0.3333
MC6.stg02
1.0000
MC6.stg03
0.1429
b) Needleman-Wunsch Maximum-Match Matrix
MC2.stg01
MC2.stg02
MC6.stg01
0.0000
0.3333
MC6.stg02
1.0000
MC6.stg03
0.1429
1.3333

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
normalised similarity, Sxy = Axy
Axx + Ayy - Axy
Sxy = =
3 + 2 –
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)
Bonds formed:
C-Cl
Bonds cleaved:
C-O
Bond order changes:
None
Overall
dipeptide epimerase (MC2)
Bonds formed:
C-H
Bonds cleaved:
Bond order changes:
None
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 D-tartrate dehydratase
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 Mechanism Similarity to
Target: o-succinylbenzoate synthase (MC1)
Mechanism Similarity to
MC1
MC2
0.5556
MC7
0.7143
dipeptide epimerase (MC2)
Kcat/KM
(M-1s-1)
E. Coli OSBS (MC1)
3.1 x 106
D297G AEE (MC2)
12.5
E323G MLE (MC7)
1.9 x 103
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 $$$ Margy Glasner Sunil Ojha Shoshana Brown
Patricia Babbitt
Noel O’Boyle
John Mitchell
Gemma Holliday
Janet Thornton
$$$
NIH
NSF
ISCB

31. Structure-Function Linkage Database
Questions?
Structure-Function Linkage Database