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Discovery and Analysis of Novel Biochemical Transformations

Discovery and Analysis of Novel Biochemical Transformations. Linda J. Broadbelt. Department of Chemical and Biological Engineering Northwestern University Evanston, IL 60208. www.clemson.edu/edisto/ corn/corn.htm. How Can We Create Products from Natural Resources?.

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Discovery and Analysis of Novel Biochemical Transformations

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  1. Discovery and Analysis of Novel Biochemical Transformations Linda J. Broadbelt Department of Chemical and Biological Engineering Northwestern University Evanston, IL 60208

  2. www.clemson.edu/edisto/ corn/corn.htm How Can We Create Products from Natural Resources? •Concern over dwindling petroleum-based resources sparks explorationof alternative feedstocks •Biochemical processes are being explored as alternatives to traditionalchemical processes www.timberland.com/.../ tim_product_detail.jsp?OID=18298 Overall reaction biomass polysaccharides monosaccharides glucose ethanol

  3. •Reactants, intermediatesand products DG3 DG2 DG1 k3 k2 k1 DG8 DG6 DG7 DG5 k8 •Reactions k6 k7 k5 DG4 DG11 k4 k11 •Thermodynamic parameters DG12 DG13 DG10 k12 k13 DG14 k10 k14 DG9 k9 •Kinetic parameters DG18 DG15 k18 k15 DG17 DG16 k16 k17 Reaction Networks of Novel Biochemical Transformations

  4. Challenges for Reaction Network Development Reactive intermediates have not been detected Pathways have not been elucidated experimentally Thermodynamic and kinetic parameters are unknown Reaction networks are large Construction is tedious and prone to user’s bias and errors Computer generation of reaction networks

  5. Elements of Computer Generated Reaction Networks • Graph Theory • Reaction Matrix Operations • Connectivity Scan • Uniqueness Determination • Property Calculation • Termination Criteria DG3 DG2 DG1 k3 k2 k1 DG8 Reactants Reaction Types Reaction Rules DG6 DG7 DG5 k8 k6 k7 k5 DG4 DG11 k4 k11 DG12 DG13 DG10 k12 k13 DG14 k10 k14 DG9 k9 DG18 DG15 k18 k15 DG17 DG16 k16 k17

  6. C 0 2 1 0 0 1 C 0 1 1 1 1 C 2 0 0 1 1 0 H 1 0 0 0 0 H 1 0 0 0 0 0 H 1 0 0 0 0 H 0 1 0 0 0 0 C 1 1 1 1 C 1 1 1 1 H 1 0 0 0 0 H 0 1 0 0 0 0 H 1 0 0 0 H 1 0 0 0 H 1 0 0 0 0 0 H 1 0 0 0 0 H 1 0 0 0 H 1 0 0 0 H 1 0 0 0 Bond-Electron Representation Allows Implementation of Chemical Reaction methane methyl radical ethylene ij entries denote the bond order between atoms i and j ii entries designate the number of nonbondedelectrons associated with atom i

  7. C 0 1 1 1 1 H 1 0 0 0 0 H 1 0 0 0 0 H 1 0 0 0 0 H 1 0 0 0 0 0 -1 1 H 0 0 1 -1 1 0 C• 0 1 0 1 0 -1 H 1 0 0 Chemical Reaction as a Matrix Addition Operation Reaction Operation H • + CH4 •CH3 + H2 H 0 1 0 + C 1 0 0 H• 0 0 1 Reactant Matrices Reordered Reactant Matrix Product Matrix Reactant Matrix H 0 1 0 0 0 0 C 0 1 1 1 1 0 H 0 0 1 0 0 0 C 1 0 0 1 1 1 H 1 0 0 0 0 0 C• 0 1 0 1 1 1 H 1 0 0 0 0 0 H• 0 0 1 0 0 0 H 1 0 0 0 0 0 H 1 0 0 0 0 0 H 0 1 0 0 0 0 H 0 1 0 0 0 0 H 1 0 0 0 0 0 H 0 1 0 0 0 0 H 0 1 0 0 0 0 H• 1 H• 0 0 0 0 0 1 H 0 1 0 0 0 0 H 0 1 0 0 0 0

  8. Unique Patterns ofObserved Enzyme Chemistry EC i.j.k.l → unique enzyme i → main class j → functional group k → cofactor / cosubstrate Tipton, S.B. and Boyce, S. Bioinformatics. 16 (2000), 34-40 Kanehisa, M. and Goto, S. Nucleic Acid Research. 28 (2000), 27-30

  9. 4th level is specific to substrate

  10. Formulation of Reaction Matrices Using Enzyme Classification System Enzyme commission (EC) code number provides systematic names for enzymes EC i.j.k.lunique enzyme ithe main class jthe specific functional groups kcofactors lspecific to the substrates

  11. Formulation of Reaction Matrices Using Enzyme Classification System Enzyme commission (EC) code number provides systematic names for enzymes EC i.j.k.lunique enzyme ithe main class jthe specific functional groups kcofactors lspecific to the substrates

  12. Generalized Enzyme Function Examined at the i.j.k Level • More than 5,000 specific enzyme functions (i.j.k.l) • Fewer than 250 generalized enzyme functions (i.j.k) • Novel enzyme functions should be expected through genomic sequencing, proteomics and protein engineering

  13. • EC 4.2.1.2 ( fumarate hydratase ) H - C - C - O - H + H O C=C 2 O H H Example of a Generalized Enzyme Reaction Generalized enzyme reaction O H (EC 4.2.1) C O H O C O H + 2 2 H O C H O C 2 2 H H H - - C - - C - - O - - H • EC 4.2.1.3 ( aconitate hydratase ) H O C O H + H O + C O H C=C 2 2 H O C H O C 2 2 2 C O H C O H 2 2

  14. O O O C C C O O O H H H 2 2 2 H H H H H H H H H O O O C C C 2 2 2 O O H H C C O O H H 2 2 H H O O C C 2 2 Matrix Representation of Generalized Enzyme Function (i.j.k) Generalized enzyme reaction EC 4.2.1 + + HOH + C=C - - - - H C C O H O C C O H C C H H C C O 0 0 0 1 0 -1 0 1 0 1 0 0 H 0 0 2 0 H H -1 0 1 0 1 0 1 0 = + C 0 2 0 0 C C 0 1 0 1 0 1 0 -1 C 1 0 0 4 C C 0 0 1 4 1 -1 0 0 O O O Reactant Products Reaction operator

  15. Discovery of Novel Biosynthetic Routes I.J.K L.M.N Q.R.S D E A + B C I.J.K L.M.N Q.R.S I.J.K L.M.N Q.R.S I.J.K L.M.N Q.R.S C C + A + B A + B C + A + B D D E Generation 0 Generation 1 Generation 2 Generation 3

  16. Implications for Novel Pathway Development Given a novel reaction (reactant/product), can we identify enzymes (catalysts) that could be engineered (evolved) to carry this novel biotransformation ? If A gives B under 2.4.1 action,then target enzymes within the 2.4.1 class

  17. Application of Reaction Matrix Approach Step 1 Enumerate all enzymes in the EC system Step 2 Choose a specific pathway to explore its synthetic ability Example Aromatic amino acid biosynthesis Exists in higher plants and microorganisms Pathway does not exist in mammals

  18. Aromatic Amino Acid Biosynthesis: Phenylalanine and Tyrosine phenylpyruvate chorismate glutamate phenylalanine prephenate dehydratase aromatic aminotransferase chorismate mutase 4-hydroxyphenylpyruvate prephenate dehydrogenase tyrosine glutamate prephenate

  19. Aromatic Amino Acid Biosynthesis: Phenylalanine and Tyrosine phenylpyruvate chorismate glutamate 2.6.1.57 phenylalanine prephenate dehydratase 5.4.99.5 aromatic aminotransferase 4.2.1.51 chorismate mutase 4-hydroxyphenylpyruvate 1.3.1.12 2.6.1.57 prephenate dehydrogenase tyrosine glutamate prephenate

  20. 4.2.1.51 + H2O + CO2 prephenate dehydratase It is both a carboxy-lyase (4.1.1)and a hydro-lyase (4.2.1) Reaction Misclassification (?) Some reactions within classes are not general General 4.2.1 reaction (4. = lyase)Loses water (4.2.1 = hydrolyase) AND forms a double bond. However…

  21. Reaction Decomposition 4.2.1.51 + H2O + CO2 prephenate dehydratase 4.2.1.51 can be broken down into 3 general reactions: 4.1.1 will decarboxylate (4.1.1 is a carboxy-lyase) 5.3.3 will rearrange the double bond (5.3.3 transposes C=C bonds) 4.2.1 will lose H2O and form a double bond (4.2.1 is a hydro-lyase)

  22. Mapping Results • Although only 2500 reactions in the KEGG and 269 reactions in the iJR904 model were contained in the curated EC classes, 3267 (50%) of the KEGG reactions and 430 (46%) of the iJR904 reactions were reproduced using the 86 reaction rules • The reproduced reactions are involved in 129 different third-level enzyme classesin the KEGG and iJR904 • 100% of the reactions contained in 25 of the uncurated EC classes in the KEGG were mapped to the 86 existing reaction rules

  23. Tryptophan Biosynthesis Pathway Input Molecules phosphoenolpyruvate (PEP), erythrose-4-phosphate (E4P), glutamine, serine, ribose-5-phosphate (R5P) Cofactors ATP, NADPH Specific Enzyme Actions 12

  24. The Evolution and Wealth of the Aromatic Amino Acid Biochemistry 105 104 TRP PHE 103 Convergence Number of Products TYR 102 10 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Generation Number

  25. Specialty Organic Chemicals 7-carboxyindole

  26. 3-Hydroxypropanoate from Pyruvate • 3-Hydroxypropanoate is a useful chemical with known biochemical production routes • Generate all of the possible compounds and reactions from pyruvate using only the reaction rules involved in the known biosynthetic routes to 3HP • Generate all of the possible compounds and reactions from pyruvate using all of the 86 current reaction rules

  27. Novel Biosynthetic Pathways Discovered: Pyruvateto 3HP Distribution of lengths of pathways 106 pathways discovered using only the reaction rules involved in the known pathways to 3HP 105 104 Number of pathways 103 102 pathways discovered using all reaction rules 10 1 2 3 4 5 6 7 8 9 10 Pathway length A pathway of length two and a pathway of length three were both discovered using the additional reaction rules

  28. 3HP-CoA Propan-2-ol Propane-1,3-diol 3-HP Acryloyl-CoA 3-hydroxypropanol Propenoate Propene Allyl alcohol Lactoyl-CoA 3-Oxopropionyl-CoA 2-hydroxy-2,4-pentadienoate Lactate Propane 1,2 diol Propane-1-ol Ethylamine Ethanol Acetaldehyde Malonate semialdehyde Ethylene Propanoyl-CoA Alanine Malate Fumarate Propanoate Aspartate Pyruvate Beta-alanyl-CoA Propanoate Oxaloacetate Beta-alanine Homoserine Theonine Acrolein Lactaldehyde Hydroxyacetone

  29. 3HP-CoA 3-HP Acryloyl-CoA Lactoyl-CoA Lactate Malonate semialdehyde Aspartate Pyruvate Beta-alanyl-CoA Oxaloacetate Beta-alanine

  30. What Screening Methods Can We Use to Identify the Most Attractive Pathways? • Pathway length • Fewest novel intermediates • Thermodynamic feasibility • Maximum achievable yield to 3HP from glucose during anaerobic growth • Maximum achievable intracellular activity at which 3HP can be produced • Protein docking calculations • Quantum chemical investigations

  31. Shortest Novel Pathways to 3HP CO2 glu 2-oxo Pyruvate Oxaloacetate Aspartate glu 4.1.1 Rev 2.6.1 Pathway N1 2-oxo H+ CO2 glu 2.6.1 2-oxo nadh 1.1.1 Alanine 4.1.1 Ethylamine Acetaldehyde CO2 4.1.1 2.6.1 4.3.1 CO2 nad NH3 4.2.1 Rev 4.3.1 Propenoate Lactate Β-alanine H2O NH3 Pathway N2 CoA 2-oxo 2.3.1 2.3.1 4.1.1 Rev 2.6.1 CoA H2O CoA H2O glu Malonate Semialdehyde Β-alanyl CoA 4.2.1 Rev Lactoyl CoA 2.3.1 H2O H2O 2-oxo 1.1.1 & 2.3.1 CoA H+ H+ glu nadh nadh 4.3.1 2.6.1 nad 4.2.1 H+ 1.1.1 3-oxopropionyl CoA nadh H2O NH3 nad nad 1.1.1 H2O CoA H2O Acryloyl CoA 3-HP-CoA 3-HP 2.3.1 Rev 4.2.1 Rev KEGG Reaction not in Patented Pathways Not found in KEGG or Patented Pathways Part of the Patented Pathway

  32. Attractive Novel Pathways Successfully Identified • Two-step pathway identified with only one novel reaction • Maximum achievable yield to 3HP from glucose during anaerobic growth matches commercial pathway • Slightly reduced maximum achievable intracellular activity at which 3HP can be produced • Numerous other attractive candidates

  33. Are These Novel Reactions Feasible? Decarboxylation reaction of ketoacids PFOR (1.2.7.a) : pyruvate + CoA + Fd (ox) CO2+ acetyl-CoA + Fd (red) Generalized enzyme operators can act on all of the above keto acids to give their corresponding products Can the enzyme that catalyzes decarboxylation of pyruvate perform catalysis of different substrates?

  34. Explore Novel Reactions Using Molecular Modeling • Substrate binding • Docking analysis • Ability to form initial enzyme-substrate bound species with no distortion to the active site of the enzyme or the cofactor • QM/MM structural studies • Follow the reaction pattern of the native substrate • Study of reaction mechanism using QM methods

  35. Enzyme Docking Results Scored using GLIDE

  36. Enzyme Docking Poses 1 2 pyruvic acid 3 2-ketobutyric acid 2-ketoisovaleric acid 4 5 6 2-ketovaleric acid 2-keto-3-methylvaleric acid 2-keto-4-methylpentanoic acid

  37. Binding Using Quantum Mechanics/Molecular Mechanics MM part : 50 Å of active site and solvent molecules ~20,000 atoms QM part : 63 atoms Geometry : B3LYP/6-31G*

  38. Comparison of Bound Structures of Different Acids: QM/MM pyruvic acid 2-ketoisovaleric acid 2-keto-3-methylvaleric acid 2-keto-4-methylpentanoic acid QM/MM structural studies suggest that the binding of the substrates does not cause distortions to the active site

  39. Kinetics of Enzyme-Catalyzed Decarboxylation: Quantum Mechanics LThDP TS1 TS2 HEThDP enamine ThDP ylide + KA Ville et al., Nature Chemical Biology, 2(6), 2006, 324

  40. Free Energy Surface of Thiamine-Catalyzed Decarboxylation: Pyruvic Acid TS 1 TS 2 ThDP+pyruvic acid LThDP enamine+ CO2

  41. Comparison of Thiamine-Catalyzed Decarboxylation Free energy barrier (∆Gactivation298K, DCE)

  42. H O C O H 2 H O O C O H H O O H C O H 2 2 N O H H 1,3,4,5 - Tetrahydroxy - Cyclohexanecarboxylic acid 3 - [1 - Carboxy - 2 - (1,4 - dihydro - pyridin - 3 - yl) - ethoxy] - 4 - hydroxy - cyclohexa - - 1,5 - dienecarboxylic acid Exploring Novel Pathways and Molecules New routes tobioavailable species New molecules Present in KEGG(Kyoto Encyclopedia of Genes and Genomes) NOT present in KEGG NOT present in CAS REGISTRY

  43. C O H 2 H O H O O H O Migration to Biocatalytic Processes New biochemical routesto existing chemicals 1,3,5 - Trihydroxy - 4 - oxo - cyclohexane carboxylic acid NOT present in KEGG Present in CAS REGISTRY

  44. Acknowledgments Funding • Department of Energy • National Science Foundation Cyber-enabled Discoveryand Innovation Collaborators • VassilyHatzimanikatis • Chunhui Li • Chris Henry • GoranKrilov • Raj Assary

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