The Chemistry of Protein Catalysis John Mitchell

The MACiE Database Mechanism, Annotation and Classification in Enzymes. http://www.ebi.ac.uk/thornton-srv/databases/MACiE/ Gemma Holliday, Daniel Almonacid, Noel O’Boyle, Janet Thornton (EBI), Peter Murray-Rust, Gail Bartlett (EBI), James Torrance, John Mitchell G.L. Holliday et al., Nucl. Acids Res., 35, D515-D520 (2007)

Enzyme Nomenclature and Classification EC Classification Class Subclass Sub-subclass Serial number

The EC Classification Only deals with overall reaction Reaction direction arbitrary Cofactors and active site residues ignored Doesn’t deal with structural and sequence information However, it was never intended to do so

A New Representation of Enzyme Reactions? Should be complementary to, but distinct from, the EC system Should take into account: Reaction Mechanism Structure Sequence Active Site residues Cofactors Need a database of enzyme mechanisms

Mechanism, Annotation and Classification in Enzymes. MACiE Database Mechanism, Annotation and Classification in Enzymes. http://www.ebi.ac.uk/thornton-srv/databases/MACiE/

Coverage of MACiE Representative – based on a non-homologous dataset, and chosen to represent each available EC sub-subclass.

Coverage of MACiE Structures exist for: 6 EC 1.-.-.- 56 EC 1.2.-.- MACiE covers: 6 EC 1.-.-.- 53 EC 1.2.-.- 156 EC 1.2.3.- 199 EC 1.2.3.4 1312/184~7 Representative – based on a non-homologous dataset, and chosen to represent each available EC sub-subclass.

Repertoire of Enzyme Catalysis G.L. Holliday et al., J. Molec. Biol., 372, 1261-1277 (2007) G.L. Holliday et al., J. Molec. Biol., accepted (2009)

Repertoire of Enzyme Catalysis Enzyme chemistry is largely nucleophilic

Repertoire of Enzyme Catalysis Enzyme chemistry is largely nucleophilic

Repertoire of Enzyme Catalysis Proton transfer AdN2 E1 SN2 E2 Radical reaction Tautom. Others

Repertoire of Enzyme Catalysis

Repertoire of Enzyme Catalysis

Repertoire of Enzyme Catalysis

Repertoire of Enzyme Catalysis

Residue Catalytic Propensities

Residue Catalytic Functions

We use a combination of bioinformatics & chemoinformatics to identify similarities between enzyme-catalysed reaction mechanisms

Just like sequence alignment! … we align the steps of chemical reactions. Just like sequence alignment! We can measure their similarity …

Find only a few similar pairs

Identify convergent evolution

Check MACiE for duplicates

Mechanistic similarity is only weakly related to proximity in the EC classification

EC in common  0 -.-.-.-  1 c.-.-.-  2 c.s.-.-  3 c.s.ss.-

Evolution of Enzyme Function D.E. Almonacid et al., to be published

EC is our Functional Classification Chemical reaction Enzyme Commission (EC) Nomenclature, 1992, Academic Press, San Diego, 6th Edition

Enzyme catalysis databases G.L. Holliday et al., Nucleic Acids Res., 35, D515 (2007) S.C. Pegg et al., Biochemistry, 45, 2545 (2006) N. Nagano, Nucleic Acids Res., 33, D407 (2005)

Coverage of MACiE Representative – based on a non-homologous dataset, and chosen to represent each available EC sub-subclass.

Based on a few evolutionarily related families Coverage of SFLD Based on a few evolutionarily related families

But without mechanisms. Coverage of EzCatDB But without mechanisms.

Work with domains - evolutionary & structural units of proteins. Map enzyme catalytic mechanisms to domains to quantify convergent and divergent functional evolution of enzymes.

CATH is our Structural Classification Orengo, C. A., et al. Structure, 1997, 5, 1093

Results: Convergent Evolution Numbers of CATH code occurrences per EC number c.-.-.- c.s.-.- c.s.ss.- c.s.ss.sn C 3.17 1.73 1.38 1.11 A 11.00 3.27 1.93 1.60 T 28.00 4.89 2.24 1.19 H 38.33 5.80 2.46 1.22 2.46 CATH/EC reaction Convergent Evolution

Numbers of CATH code occurrences per EC number Results: Convergent Evolution Numbers of CATH code occurrences per EC number c.-.-.- c.s.-.- c.s.ss.- c.s.ss.sn C 3.17 1.73 1.38 1.11 A 11.00 3.27 1.93 1.60 T 28.00 4.89 2.24 1.19 H 38.33 5.80 2.46 1.22 2.46 CATH/EC reaction: Convergent Evolution An average reaction has evolved independently in 2.46 superfamilies

Results: Divergent Evolution database entries/CATH EC reactions/CATH C 4.75 19.50 39.25 90.00 A 3.14 7.00 10.48 17.90 T 1.36 1.79 2.08 3.05 H 1.20 1.36 c.-.-.- c.s.-.- c.s.ss.- c.s.ss.sn 1.46 2.05 1.46 EC reactions/CATH Divergent Evolution database entries/CATH 2.18

Results: Divergent Evolution database entries/CATH EC reactions/CATH C 4.75 19.50 39.25 90.00 A 3.14 7.00 10.48 17.90 T 1.36 1.79 2.08 3.05 H 1.20 1.36 c.-.-.- c.s.-.- c.s.ss.- c.s.ss.sn 1.46 2.05 1.46 EC reactions/CATH: Divergent Evolution An average superfamily has evolved 1.46 different reactions database entries/CATH 2.18

The Future …

(1) Molecular Evolution

Now we want to evolve chemical reactions in silico across chemical, or EC, space. 1. To understand and rationalise convergent and divergent biochemical evolution; 2. To better relate protein structure and function; 3. To understand the influence on networks of coupled reactions.

(2) Understanding Protein Structure We seek to understand the influence of folding pathway on protein structure over all time scales (including the evolutionary one).

5788 (~12%) of PDB come from Structural Genomics 44

Protein Folding Funnel Energy Landscape 5788 (~12%) of PDB come from Structural Genomics 45

ACKNOWLEDGEMENTS Dr Gemma Holliday Dr Daniel Almonacid Dr Noel O’Boyle Prof. Janet Thornton (EBI) Dr Peter Murray-Rust Dr Florian Nigsch

ACKNOWLEDGEMENTS Cambridge Overseas Trust