Classification of Mechanistically Diverse Enzyme Superfamilies
According to Similarities in Reaction Mechanism
Daniel E. Almonacid and Patricia C. BabbittDaniel E. Almonacid and Patricia C. Babbitt18th July 2008
OverviewOverviewIntroduction
MotivationE.C. ClassificationEnzyme Catalysis DatabasesStructure-Function Linkage Database
MethodsEnolase SuperfamilyComputing Similarity of Mechanisms and Overall Reactions
ResultsOverall vs Mechanism SimilarityComplete Linkage ClusteringApplications
Conclusions
MotivationMotivation
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.
E.C. ClassificationE.C. Classification
Enzyme Commission (EC) Nomenclature, 1992, Academic Press, 6th Ed.
Enzyme Catalysis DatabasesEnzyme Catalysis Databases
Holliday, G. L., et al. Nucleic Acids Res., 2007, 35, D515
Pegg, S. C.-H., et al. Biochemistry, 2006, 45, 2545
SFLD (SFLD (http://sfld.rbvi.ucsf.edu/))
Enolase SuperfamilyEnolase Superfamily
Enolase SuperfamilyEnolase Superfamily
Enolase SuperfamilyEnolase Superfamily
Enolase SuperfamilyEnolase Superfamily
Enolase SuperfamilyEnolase Superfamily
Enolase SuperfamilyEnolase Superfamily
Enolase SuperfamilyEnolase Superfamily
DatasetDatasetLabelingE1GD1MR1...
Computing Mechanism SimilarityComputing Mechanism Similarity
dipeptide epimerase (MC2)
chloromuconate cycloisomerase (MC6)
dipeptide epimerase (MC2)
chloromuconate cycloisomerase (MC6)
Step 3
Bonds formed:• None
Bonds cleaved:• C-O
Bond order changes:• C-O C=O• C=C C-C• C-C C=C
Step 2
Bonds formed:• C-H
Bonds cleaved:• Base-H
Bond order changes:• C-O C=O• C=C C-C
Step similarity (Tanimoto coeff) = intersection / union = 2/(4+4-2) = 0.3333
Similarity between Reaction StepsSimilarity between Reaction Steps
Global Alignment of Reaction SequencesGlobal Alignment of Reaction Sequences
MC2.stg01 MC2.stg02MC6.stg01 0.0000 0.3333MC6.stg02 1.0000 0.0000MC6.stg03 0.1429 0.3333
MC2.stg01 MC2.stg02MC6.stg01 0.0000 0.3333MC6.stg02 1.0000 0.0000MC6.stg03 0.1429 1.3333
b) Needleman-Wunsch Maximum-Match Matrix
a) Similarity Matrix
Step 1
Step 2
Step 3
Step 1
Step 2
1.0
0.3333
normalised similarity, Sxy = Axy
Axx + Ayy - Axy
Alignment score, Axy, of 1.3333
NM O’Boyle, et al., J. Mol. Biol., 2007, 368, 1484.
Similarity between Reaction MechanismsSimilarity between Reaction Mechanisms
Sxy = 1.3333 = 0.3636
3 + 2 – 1.3333
chloromuconate cycloisomerase
(MC6)
dipeptide epimerase
(MC2)
Overall Overall vsvs Mechanistic Similarity 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.
dipeptide epimerase (MC2)
chloromuconate cycloisomerase (MC6)
Similarity between Overall ReactionsSimilarity between Overall Reactions
dipeptide epimerase (MC2)
chloromuconate cycloisomerase (MC6)
Overall
Bonds formed:• C-Cl
Bonds cleaved:• C-O
Bond order changes:• None
Overall
Bonds formed:• C-H
Bonds cleaved:• C-H
Bond order changes:• None
Overall similarity (Tanimoto coeff) = intersection / union = 0/(4+4-0) = 0
Similarity between Overall ReactionsSimilarity between Overall Reactions
Overall Overall vsvs Mechanistic Similarity 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.
R-O
O
HBase-
Common partial reaction:
chloromuconate cycloisomerase (SFLD10)
dipeptide epimerase (SFLD12)
Complete Linkage Clustering of MechanismsComplete Linkage Clustering of Mechanisms
R-O
O
HBase-
Common partial reaction:
chloromuconate cycloisomerase (SFLD10)
dipeptide epimerase (SFLD12)
Same Subgroup, Different MechanismSame Subgroup, Different Mechanism
chloromuconate cycloisomerase
dipeptide epimerase
R-O
O
HBase-
Common partial reaction:
D-tartrate dehydratase
Enolase
o-succinyl- benzoate synthase
Different Subgroup, Same MechanismDifferent Subgroup, Same Mechanism
enolase (E1)
D-tartrate dehydratase (MR1)
o-succinylbenzoate synthase (MC1)
Different Subgroup, Same MechanismDifferent Subgroup, Same Mechanism
R-O
O
HBase-
Common partial reaction:
Preliminary Anecdotal ObservationPreliminary Anecdotal Observation
o-succinyl- benzoate synthase
muconatecycloisomerase
dipeptide epimerase
muconate cycloisomerase (MC7)
dipeptide epimerase (MC2)
Schmidt, et al., Biochemistry, 2003, 42, 8387.
Mechanism Similarity to
MC1MC2 0.5556
MC7 0.7143
Target: o-succinylbenzoate synthase (MC1)
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
Preliminary Anecdotal ObservationPreliminary Anecdotal Observation
ConclusionsConclusions 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
AcknowledgementsAcknowledgements
Noel O’BoyleJohn Mitchell
Gemma HollidayJanet Thornton
Margy GlasnerSunil OjhaShoshana BrownPatricia Babbitt
$$$NIHNSFISCB
Questions?Questions?
Structure-Function Linkage Database
http://sfld.rbvi.ucsf.edu/
