Daniel E. Almonacid and Patricia C. Babbitt

transcript

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

DatasetDatasetLabelingE1GD1MR1...

Computing Mechanism SimilarityComputing Mechanism Similarity

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

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

dipeptide epimerase

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.

Similarity between Overall ReactionsSimilarity between Overall Reactions

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.

HBase-

Common partial reaction:

chloromuconate cycloisomerase (SFLD10)

dipeptide epimerase (SFLD12)

Complete Linkage Clustering of MechanismsComplete Linkage Clustering of Mechanisms

HBase-

chloromuconate cycloisomerase (SFLD10)

dipeptide epimerase (SFLD12)

Same Subgroup, Different MechanismSame Subgroup, Different Mechanism

chloromuconate cycloisomerase

dipeptide epimerase

HBase-

D-tartrate dehydratase

Enolase

o-succinylbenzoate 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

HBase-

Preliminary Anecdotal ObservationPreliminary Anecdotal Observation

o-succinylbenzoate synthase

muconatecycloisomerase

dipeptide epimerase

muconate cycloisomerase (MC7)

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)

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?

daniel.almonacid@ucsf.edu

Structure-Function Linkage Database

http://sfld.rbvi.ucsf.edu/

Daniel E. Almonacid and Patricia C. Babbitt

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