Instantiation of Generic Reactions

1. Instantiation of Generic Reactions by Markus Krummenacker Q1 2015

2. Generic Reactions • Some enzymes have broad substrate specificity • Therefore, many EC reactions are formulated as generic reactions, with some compound classes as substrates. Often, the full specificity range is unknown. • A generic reaction is a more compact representation than listing every instance reaction explicitly • BioCyc PGDBs have many generic reactions • Examples: • 1.1.1.69: NAD(P)+ + D-gluconate = NAD(P)H + 5-dehydro-D-gluconate + H+ • 1.3.99.3: a 2,3,4-saturated fatty acyl CoA + FAD -> FADH2 + a 2,3-dehydroacyl-CoA

3. Problem with FBA • The reaction network in FBA models is formulated in terms of specific compound instances • Problem: disconnect between class and instance frames • Example (to eventually produce cardiolipin): • D-glyceraldehyde-3-phosphate + phosphate + NAD+ -> 1,3-bisphospho-D-glycerate + H+ + NADH • dihydroxyacetonephosphate + NAD(P)H + H+ -> sn-glycerol-3-phosphate + NAD(P)+ • Remedy: automatically generate instance-based versions from a generic reaction • Runs as a preprocessing step. Instance reactions are not saved to the PGDB.

4. Cases of Generic Reactions • Individual generic reactions: can be part of pathways or be standalone. The most common case. • Polymerization pathways: a series of reactions needs instantiation for several cycles. • Single polymerization reactions: like glycogen metabolism. Not handled currently. • The success rate of instantiation depends on how thoroughly a PGDB was curated. There are still many generic reactions for which this does not work well.

5. Instantiation Algorithm • Generic reaction: |Xs| + H2O = |Ys| • |Xs| is a class with instances X1 X2 X3 • |Ys| is a class with instances Y1 Y2 • Instantiation code tries to pair all instances on LEFT and RIGHT sides with each other, substituting for the class, leading to temporary reactions like: • X1 + H2O = Y1 • X2 + H2O = Y1 • X1 + H2O = Y2 etc. • Test whether for a given instance in |Xs| , there is only 1 instance in |Ys| that leads to a mass-balanced reaction equation. If yes, create the instance-based reaction on the fly. • (No chemical structure matching yet.) • If an existing reaction frame for the instance based reaction can be found, it is used instead of the instantiation.

6. Requirements • Reactions have to be fully mass balanced • Compound instances need to be created, with structures • Compound structures need pH7.3 protonation • Compound instances have to be correctly classified under the classes used in generic reactions • Right-click command Edit->Compound Editor • Multiple instances with identical chemical formula will be ambiguous

7. Practical Example • Right-click command “Show reaction’s instantiations in terminal” • EC# 1.1.1.69 • GLUCONATE-5-DEHYDROGENASE-RXN : NAD(P)+ + D-gluconate = NAD(P)H + 5-dehydro-D-gluconate + H+ [balanced] • successes: • NADP+ + D-gluconate = NADPH + 5-dehydro-D-gluconate + H+ • NAD+ + D-gluconate = NADH + 5-dehydro-D-gluconate + H+ • failures: • non-unique-balanced-instantiations (cannot decide which of several instantiations is correct): • success vs. failures vs. non-unique-balanced-instantiations: 2 / 0 / 0

8. Debugging of Pathways • Right-click command “Show pathway’s instantiated reactions in terminal” • Conveniently shows results for all reactions • Debugging: If lots of problems, it helps to put a compound into the biomass that occurs early in pathway, to see if this at least can be produced • Example pathways to instantiate: • proline biosynthesis I • L-idonate degradation

9. Special ETR Instantiation • Electron Transfer Reactions (ETRs) refer to quinone classes, usually. • Different isoprenoid tail lengths exist in various organisms. • Uses NCBI taxonomy for selection of tail length. B. subtilisuses menaquinone-7 • Default instantiation is ubiquinone-8 and menaquinone-8

10. Special Compartments Instantiation • Schema change in BioCyc 15.0 regarding representing compartments of reactions • Now, 1 reaction can be assigned to multiple compartments. • FBA makes compartment-specific instantiated reactions to differentiate between the compartments

11. Syntax of Instantiation IDs • Every instantiated reaction gets assigned a unique ID • Visible in .sol file • Constructed from the generic reaction and the IDs of the instance compounds on the left and right • Format: GEN-RXN-ID-L1/L2//R1/R2.suffix-len. • Non-default compartments: • GEN-RXN-ID[CCO-PERI-BAC]-L1/etc….

12. Polymerization Pathways • Cyclic pathways of generic reactions

13. Polymerization Pathway Instantiation • A series of instantiated reactions is needed to reach a product of a certain length • Run cycle for 8 iterations (hard-coded, for now) • Structures of class compounds have R groups • The hallmark of a polymerization pathway is that 1 reaction, the polymerization step, is unbalanced. • For now, the chemical formula of the misbalance is determined, which stands for the monomer unit. (No structural information is used, yet.) • Appropriate instance compounds are searched by replacing the R groups with an integral multiple of the misbalance • Still a bit experimental.

14. Instantiated reactions in .sol • In the .sol file, instantiated reactions are listed in full detail, in the reactions sections • In the .dat file, to be used for the Cellular Overview, fluxes of instantiated reactions are all combined into a value for the base generic reaction, because the Cellular Overview can only show the latter.