1. MACiE – a Database of Enzyme Reaction Mechanisms Janet Thornton EMBL-EBI July 2006

2. Enzymes in Data Resources (2005) No. enzymes Total No. %-tage in DB UniProt 65,076 172,690 37.7 PDB (all entries) 14,143 31,522 44.9 PDB (non-redundant) 3,655 10,450 35.0 Reactome (human) (via UniProt) 230 680 33.8 Roman Laskowski

3. Relating the number of enzymes to proteome size Proteome size Number of enzymes Permissive set KEGG assignments Conservative set human mouse worm fly Shiri Freilich

4. Increase in Number of Different Reactions (E.C.) in larger proteomes E.C. 1 E.C. 2 E.C. 3 E.C. 4 E.C. 5 E.C. 6 Freilich et al (2005) JMB ??

5. Extension and Evolution of Pathways: The integration of the steroid biosynthesis pathway into the sterol biosynthesis pathway sterol cholesterol steroid hormone bile acid Universal metazoa human Shiri Freilich

6. Enzyme Structure, Function and Evolution Outstanding Research Questions: –How is catalysis performed Principles of catalysis? E.C. numbers –How do enzymes evolve? –Can we predict enzyme function from structure? –Can we design new enzymes? –What is the enzyme complement in different organisms and how does it evolve? Trypsin Need a list of active sites

7. What constitutes a catalytic residue? Direct involvement in the reaction mechanism Polarises or alters the pK a of a residue or water molecule which is directly involved in the reaction mechanism Polarises or activates part of the substrate (e.g. making a bond more susceptible to cleavage) Stabilisation of a transition-state intermediate

8. http://www.ebi.ac.uk/thornton-srv/databases/CSA The Catalytic Site Atlas: a resource of catalytic sites and residues identified in enzymes using structural data. Porter, Bartlett, & Thornton Nucl. Acids. Res. (2004) 32: D129-D133.  -lactamase Class A; EC 3.5.2.6; PDB: 1btl –Reaction:  -lactam + H 2 O   -amino acid –Active site residues: S70, K73, S130, E16

9. Comparison of CSA, SwissProt & PDB (2004) Porter, Bartlett et al, 2004 NAR

10. Conformational Change Templates Spherical Harmonics Binding Site Diversity Metabolome Ligand Selectivity Catalytic Site Atlas

11. CSA Coverage and Annotations Generated Current entries in CSA from Literature = 737 Current proteins in UniProt annotated by homology = 14,863 Functional annotations by homology are more accurate if catalytic residues are checked and conserved (George et al (2005) PNAS) BUT no possibility of storing or querying the proposed chemical mechanisms (which must be available to identify the catalytic residues in the CSA)

12. The MACiE Database - a Research Project Mechanism, Annotation and Classification in Enzymes G. L. Holliday, G. J. Bartlett, Daniel Almonacid P. Murray-Rust, J.M.Thornton J. B. O. Mitchell (Holliday et al Bioinformatics 2005 21:4315 ) http://www.ebi.ac.uk/thornton-srv/databases/MACiE

13. Why develop MACiE? To understand more about catalysis –To gather information on mechanisms –To compare and contrast mechanisms in different proteins –To help validate enzyme mechanisms –To study the evolution of mechanisms –To develop mechanism-based classification of enzymes –To help predict mechanism from structure –To help design new enzymes

14. http://www.ebi.ac.uk/thornton-srv/databases/MACiE The MACiE Database

15. Content in MACiE ● Enzyme Name: fructose-bisphosphate aldolase ● E.C. Classification: (EC 4.1.2.13) – Obsolete EC codes associated with entry: EC 4.1.2.7 ● Reference Structure: PDB 1b57 – Domain classification: CATH 3.20.20.70 – UniProt code: P11604 – Specie: Escherichia coli (Bacteria) – Cofactors: Zn2+ and Na+ – Catalytic residues: Asp109, Glu182, Asn286 ● Links

16. Classifying Residue Catalytic Function Hydrogen Donor, Hydrogen bond acceptor, Proton Relay Nucleophile, Electrophile Radical relay, Hydride relay Radical Donor, Radical stabiliser Leaving group, Steric role, Charge stabiliser Covalently attached, Metal ligand

17. Overall Reaction fructose-bisphosphate aldolase glycerone phosphate D-glyceraldehyde 3-phosphate D-fructose 1,6-bisphosphate +

18. Step Annotation in MACiE Step 1: Reactants

19. Step 1: Mechanism

20. Step 1: Cofactors

21. Step 1: Spectator Residues

22. Step 1: Reactant Residues

23. Similarly Step 2 is annotated Where the information is available the rate determining step is annotated

24. Step 3 is annotated MACiE always endeavours to return the enzyme to its ground state. This is often inferred, which is noted in the annotation

25. Finally: any spontaneous changes are included These are often spontaneous and occur outside the enzyme There is no other annotation involved in steps like this

26. Complete Reaction Annotation

27. Searching MACiE

28. General Searches ● Query MACiE by reaction comments ● Query MACiE by enzyme and species (scientific and common) names ● Query the chemical changes in MACiE ● Overall reactants and products (by KEGG and ChEBI compound id or compound name)

29. Frequencies of amino acid reactants performing a given function

30. Combining the amino acid and functional clusterings No reactant Function No strong preference for function Strong preference for acid/base function

31. Roles of catalytic residues and mechanistic steps in homologous enzymes of different function Gail Bartlett –How do enzymes modify the chemical reaction they catalyse, using the same structural scaffold? –Do catalytic residues conserve their role and / or identity in enzyme- catalysed reactions?

32. Methods 178 enzyme dataset with assigned catalytic residues and proposed mechanism PSIBLAST run against NRDB + PDB (cutoff e=10 -5 ) Twenty-seven pairs of homologous proteins of totally different function (at primary EC level) Structural alignment performed using SSM server Structurally equivalent catalytic residues Information manually extracted from literature Catalytic mechanisms Comparison of function, active site, catalytic residues and catalytic mechanism

33. Results - overview 27 pairs of proteins 3 enzyme / nonenzyme pairs 24 enzyme / enzyme pairs, from 21 enzyme superfamilies

34. Change of function (EC) class Function Y Function X EC 1EC 2EC 3EC 4EC 5EC 6Nonenzyme EC 1 (oxidoreductases) -033012 EC 2 (transferases) -21100 EC 3 (hydrolases) -3101 EC 4 (lyases) -710 EC 5 (isomerases) -01 EC 6 (ligases) -0

35. Enzyme / enzyme pairs All but one enzyme pair have their active site located at the same place in the protein fold Substrates and / or products shared by 11 pairs Cofactor shared by 5 enzyme pairs

36. Metal ion binding sites Conserved metal ion type Altered metal ion type Conserved metal ion function 50 Altered metal ion function 34 Twelve enzyme pairs conserve metal binding sites and ligands to the metal ions are structurally aligned Where the metal ion type has altered, subtle mutations to the ligand binding site have occurred

37. Rubredoxin oxygen oxidoreductase / metallo-  -lactamase Metallo-  -lactamase uses a Zn 2+ -activated hydroxyl for nucleophilic attack on the  - lactam substrate Rubredoxin oxygen oxidoreductase reduces dioxygen via a redox cycle at a di-iron site Fe ligandZn2+ ligand His 79His 82 Glu 81His 84 Asp 83Asp 86 His 146His 145 Asp 165Cys 164 His 226His 206

38. Catalytic residue pairs

39. Change in residue identity and residue function Endonuclease IV / Xylose isomerase DNA hydrolysis Xylose  Xylulose

40. Change in residue identity and residue function Trp 136 His 109 Endonuclease IVXylose isomerase Zn 2+ -assisted hydroxyl performs nucleophilic attack on DNA backbone Base catalysed ring opening, followed by intramolecular hydride transfer and ring closure

41. Evolution of Mechanism Mechanisms share a common step at the beginning of the overall reaction pathway, catalysed by residues which are structurally equivalent in both enzymes Mechanisms share a common step somewhere in the middle of the overall reaction path, catalysed by residues which are structurally equivalent in both enzymes Mechanisms share common steps at the beginning and end of the overall reaction path, catalysed by residues which are structurally equivalent in both enzymes, but have a different step in the middle Mechanisms do not share any common steps catalysed by structurally equivalent residues 7 7 2 8 Bartlett et al JMB

42. Common first step dehydroquinate synthase / glycerol dehydrogenase a. Dehydroquinate synthase b. Glycerol dehydrogenase several stages

43. Common first step dehydroquinate synthase / glycerol dehydrogenase Superposition of Zn 2+ and ligands dehydroquinate synthase H271 E194 H287 H275 glycerol dehydrogenase H255 H252 D169 H269

44. Conclusions Enzymes are economical in their use of active site residues and features It is more likely for residues conserving function to also conserve their identity Residues not conserving function tend to mutate Tend to find common mechanistic steps at the beginning and ‘middle’ of reaction paths – possibly the most energetically difficult step or intermediate is conserved

45. Future Increase coverage in MACiE Analyse Catalytic Mechanisms –Ingold Reaction Types; effects of non-polar residues Evolution of enzymes, pathways & metabolism in different organisms & tissues Design??

46. Acknowledgements Gail Bartlett, Craig Porter, Jonathan Barker The MACiE Team – Cambridge Univ Chemistry Dept John Mitchell, Daniel Almonacid, Peter Murray-Rust BBSRC, MRC, Wellcome Trust, EMBL James Torrance Alex Gutteridge Gemma Holliday