Ambiguity and the Choice of Functional Roles

The Overall Set of Reactions

The TyrA Aspect of the Problem

I also feel that I need to add a few comments made by Roy in order to set the stage properly for this discussion:

• First, Roy feels that we should focus on the TyrA issues and settle them, since getting on to the "Stage 3" analysis was one of our original goals in starting this discussion.
• Roy also notes that the EC numbers are not particularly helpful. Even a quick perusal of the KEGG maps versus the simple table of reactions I gave earlier makes it clear that at least the use of ECs (in this case) probably obscures more than it reveals.
• If we try to do the entire subsystem that I mentioned at the start, then 80-90% of the difficulty will relate to disambiguating aminotransferases.

The Hybrid Alternative

• In the subsystem, use the four functional roles given as the second alternative, but add one more: Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity.
• For each gene that has not been disambiguated, use this new role as the function.
• For each gene that is completely disambiguated, use the second alternative expressions (based on ";" and "@").
• For a gene that has been partially characterized (e.g., it is known that it plays at least the role Arogenate dehydrogenase, NADP specific (EC 1.3.1.43) and maybe more) use the known functions followed by ; Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity. That is, the gene would match the precise functional roles, as well as the "catch-all" role we added to the spreadsheet.

Thus, we would use the following functional roles:

Functional Role
Arogenate dehydrogenase, NAD specific (EC 1.3.1.43)
Arogenate dehydrogenase, NADP specific (EC 1.3.1.43)
Prephenate dehydrogenase, NAD specific (EC 1.3.1.13)
Prephenate dehydrogenase, NADP specific (EC 1.3.1.13)
Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity

This leads to the following ways to express gene function:

Gene	Gene Function
NADTyrAa	Arogenate dehydrogenase, NAD specific (EC 1.3.1.43)
NADPTyrAa	Arogenate dehydrogenase, NADP specific (EC 1.3.1.43)
NAD(P)TyrAa	Arogenate dehydrogenase, NAD specific (EC 1.3.1.43) @ Arogenate dehydrogenase, NADP specific (EC 1.3.1.43)
xTyrAa	Arogenate dehydrogenase, NAD specific (EC 1.3.1.43) ; Arogenate dehydrogenase, NADP specific (EC 1.3.1.43); Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity
NADTyrAc	Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity
NADPTyrAc	Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity
NAD(P)TyrAp	Prephenate dehydrogenase, NAD specific (EC 1.3.1.13) @ Prephenate dehydrogenase, NADP specific (EC 1.3.1.13)
xTyrAc	Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity
NADTyrAp	Prephenate dehydrogenase, NAD specific (EC 1.3.1.13)
NADPTyrAp	Prephenate dehydrogenase, NADP specific (EC 1.3.1.13)
NAD(P)TyrAp	Prephenate dehydrogenase, NAD specific (EC 1.3.1.13) @ Prephenate dehydrogenase, NADP specific (EC 1.3.1.13)
xTyrAp	Prephenate dehydrogenase, NAD specific (EC 1.3.1.13) ; Prephenate dehydrogenase, NADP specific (EC 1.3.1.13); Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity
NADTyrAx	Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity
NADPTyrAx	Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity
NAD(P)TyrAx	Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity
xTyrAx	Cyclohexadienyl dehydrogenase of unknown substrate and cofactor specificity

These are not completely satisfactory, but I believe they are pretty close to what we want. In a few cases, information is lost. For these very few, I believe that the curator can maintain external records until things clarify.

Anyone reading the spreadsheet can get an accurate grasp of what is known with high probability and what issues remain murky.

The Issue of How Much Detail to Include in Functional Roles

A similar issue will arise as we consider the chorismate mutase, which occurs in a closely related piece of metabolism. In this case, there are three distinct, nonhomologous forms of the enzyme. The question here is Should we have a single chorismate mutase column (representing comments relating to form as either notes, annotations, or attributes), or should we have three distinct columns?" Veronika puts all three forms in a single column, while a number of us have adopted the convention of placing nonhomologous alternatives in separate columns.

Veronika feels (obviously correctly) that as the spreadsheet becomes huge, we lose an ability to maintain an overview. The detail swamps the representation of the essential.

I am exploring the issue of how well we can have both detail and overview by proper use of subsets. I will report on my experiments at the April meeting, undoubtedly using aromatic metabolism as a setting for exploring the issues.

Before leaving this topic, let us consider the position that including the cofactors in the functional role obscures the situation rather than clarifying it. We have chosen (in most cases) to leave properties like thermostable out of the functional role. Why include cofactors? Which cofactors are needed is important, but we consciously have chosen not to keep all important aspects of the function within the actual functional role. Might it not be better to save the cofactor elsewhere? I am reluctant to do so, since I would like to capture the actual reactions. On the other hand, we can attach multiple reactions to a single functional role. This view would lead to the following version:

Functional Role
Arogenate dehydrogenase (EC 1.3.1.43)
Prephenate dehydrogenase(EC 1.3.1.13)

Uncertainty or ambiguity would be represented using the ";" and "/" operators. There is a certain appeal to this brevity.

Veronika believes that there should be three functional roles: the two above and Cyclohexadienyl dehydrogenase which would cover both broad specificity or uncertainty.