1. “Creating the right chemistry” in Pathways Master Seminar Fall 2006 M.A. Ott (CMBI)

2. Main concept ”Creating the right chemistry” in Pathways M.A. Ott Enzymes don’t do new chemistry; they do ‘old’ chemistry faster Enzymes don’t perform magic; they perform known (organic) chemistry

3. Contents ”Creating the right chemistry” in Pathways M.A. Ott Enzymes and Metabolites Metabolic Pathways and Reactions Representing Chemistry BioMeta Database

4. Ongoing progess in “omics” ”Creating the right chemistry” in Pathways M.A. Ott Genomics…going strong Transcriptomics…good progress Proteomics…some progress Metabolomics…less progress Systems biology…not in ten years

5. Metabolite and pathway databases ”Creating the right chemistry” in Pathways M.A. Ott http://www.genome.ad.jp/kegg/ http://www.brenda.uni-koeln.de/ http://www.ebi.ac.uk/chebi/ http://www.expasy.org/tools/pathways/ http://www.empproject.com/ http://metacyc.org/ http://ecocyc.org/ http://www.ncgr.org/pathdb/ http://medicago.vbi.vt.edu/B-Net/ http://www.mol-net.de/biopath/ http://www.ergo-light.com/ERGO/ http://www.reactome.org/ http://www.chem.qmul.ac.uk/iubmb/enzyme/ Kyoto Encyclopedia of Genes and Genomes BRENDA enzyme database Chemical Entities of Biological Interest ExPASy biochemical pathway EMP enzymology and metabolism data MetaCyc metabolic pathways EcoCyc E. coli genome and metabolism PathDB biological pathways and networks B-NET - Biochemical Network BioPath C@ROL database ERGO comprehensive genome analysis Reactome biological processes IUBMB enzymes

6. Metabolism and friends ”Creating the right chemistry” in Pathways M.A. Ott Metabolism: The reactions in organisms Anabolism: Synthesis of larger biomolecules from smaller ones » Usually requires energy input Catabolism: Break down of larger molecules into smaller ones » Usually releases energy

7. Enzymes ”Creating the right chemistry” in Pathways M.A. Ott Virtually all metabolic reactions require enzymatic catalysis Rate enhancement factor can be up to 10 15 Enzymes are highly reaction-specific, catalyzing only one conversion Enzymes are substrate-specific, limiting their action to only one compound or to related compounds

8. The metabolic network ”Creating the right chemistry” in Pathways M.A. Ott

9. Some metabolites ”Creating the right chemistry” in Pathways M.A. Ott “Housekeeping” metabolites: Water, NADH, ATP, Phosphate, … End products of pathways: Vitamin B 12, Urea, Penicillin, Ecdysone, … Intermediates in pathways: Glucose-6-P, Lanosterol, Precorrins, Shikimate, …

10. Housekeeping metabolites: ATP / ADP ”Creating the right chemistry” in Pathways M.A. Ott Full structures: Shorthand:

11. Housekeeping metabolites: NADH / NAD + ”Creating the right chemistry” in Pathways M.A. Ott Full structures: Shorthand:

12. Housekeeping metabolites and chemical distance ”Creating the right chemistry” in Pathways M.A. Ott What is the chemical distance between B and D?

13. Typical housekeeping metabolites ”Creating the right chemistry” in Pathways M.A. Ott Water Ammonia Phosphate ATP, ADP NADH, NAD + NADPH, NADP + FAD, FADH2 FMN, FMNH2 SAM, SAH Coenzyme A Glutamate/2-Oxoglutarate Phosphorylation Reduction/oxidation Methyl group transfer Acyl group transfer Amino/ketone group exchange

14. Cholesterol biosynthesis outline Pathways and Molecules M.A. Ott

15. Cholesterol biosynthesis from squalene Pathways and Molecules M.A. Ott

16. Metabolic disorders Pathways and Molecules M.A. Ott Inability to synthesize compounds: Absence of important compounds Inability to degrade compounds: Accumulation of undesired waste products E.g., in phenylketonuria

17. Phenylketonuria (error in phenylalanine catabolism) Pathways and Molecules M.A. Ott

18. The BioMeta Database ”Creating the right chemistry” in Pathways M.A. Ott

19. The BioMeta Database ”Creating the right chemistry” in Pathways M.A. Ott

20. BioMeta database structure ”Creating the right chemistry” in Pathways M.A. Ott

21. BioMeta database structure ”Creating the right chemistry” in Pathways M.A. Ott Molecules Reaction 1: Mol_1 + Mol_2  Mol_3 + Mol_4 Reaction 2: Mol_2 + Mol_3  Mol_5 + Mol_6 Reaction 3: 2 Mol_6  Mol_7 R-M Links Reactions

22. Sample database query ”Creating the right chemistry” in Pathways M.A. Ott ec_nr | enzyme_name | rxn_id |rev | role |stoich| name --------+-----------------+----------+----+------+------+------------ 1.2.3.4 | oxalate oxidase | MR000247 | ir | s | 1 | Oxalic acid,, |,, |,, |,, | s | 1 | O2,, |,, |,, |,, | p | 1 | H2O2,, |,, |,, |,, | p | 2 | CO2

23. Molecular structure identification ”Creating the right chemistry” in Pathways M.A. Ott Mesomerism (resonance forms) Tautomerism (variable positions of hydrogens) Other “chemical” flexibility Protonation state

24. Molecular structures and reactions ”Creating the right chemistry” in Pathways M.A. Ott Reactions:balancing atoms & charges adding direction/reversibility Molecules:adding stereochemical configurations “canonicalizing” tautomeric form No stereochemistry Phosphorus imbalance Direction Carbon imbalance

25. And now what’s really going on… ”Creating the right chemistry” in Pathways M.A. Ott

26. Accuracy required! ”Creating the right chemistry” in Pathways M.A. Ott Without accurate chemical structures and balanced reaction descriptions, a biochemical pathways database is unsuitable for “chemical computation”: Graph-theoretical considerations for creating a network Idem for describing fluxes Docking applications need correct stereochemistry

27. Validation results (structures) ”Creating the right chemistry” in Pathways M.A. Ott Type of Error# in KEGG# in BioMeta# Corrected Structure missing12391106 133 Valence violation(s) 76 0 Incorrect constitutionunknown 107 Total (constitution)13151106 316 Undefined stereo double bond(s) 35 32 3 Invalid sp3 stereocenter(s) 70 47 23 Ambiguous sp3 stereocenter(s) 46 0 Undefined sp3 stereocenter(s) 1398 865 533 Unspecified enantiomer23261840 486 Undefined sp3 stereochemistry 554 366 188 Incorrect stereochemistryunknown 69 Total (stereochemistry) 399029071152 Total corrected 1468

28. Validation results (reaction balance) CountPerc. Total6089100.00 Unknown76612.58 Balanced371160.95 Unbalanced161226.47 Corrected *89314.66 ”Creating the right chemistry” in Pathways M.A. Ott DifferenceCount [H+] *801 2[H+] *35 H 2 O *57 O98 H2H2 17 H 2 – 2[H+]41 2H 2 17 H 2 – [H+]18 CH 2 30 O 2 – H 2 O17

29. Some unresolved issues ”Creating the right chemistry” in Pathways M.A. Ott

30. BioMeta database ”Creating the right chemistry” in Pathways M.A. Ott Based on freely available data (KEGG Ligand database) Augmentation, completion and correction of small-molecule information: - Adding missing stereochemistry to structures - Balancing atoms and charges in reactions - Establishing the reversibility and direction of reactions Search for alternative pathways between metabolites Search for “missing” enzymes

31. Alternative pathways ”Creating the right chemistry” in Pathways M.A. Ott

32. BioMeta: things being done ”Creating the right chemistry” in Pathways M.A. Ott Enhancing compound/reaction structures Establishing directionality/reversibility of reactions Distinguishing cofactors/“current”/“external”/ “common” metabolites from ‘real’ metabolites Adding more compound data, e.g. pK a (curve) Increasing functionality of WWW interface

33. BioMeta: things to do ”Creating the right chemistry” in Pathways M.A. Ott Atom-to-atom correspondence (reaction mapping) Tautomer handling Pathway searches Putative/hypothetical reactions for predicting metabolism

34. Acknowledgements ”Creating the right chemistry” in Pathways M.A. Ott Felix van Diggelen Gert Vriend CMBI