D756 Nucleic Acids Research, 2012, Vol. 40, Database issue Table 1. The master reaction RHEA:15133 (shown in Figure 3) has an undeﬁned direction, represented with the symbol <?>. It is associated with three directional reactions (RHEA:15134, RHEA:15135 and RHEA:15136), each of which has a speciﬁc set of corresponding cross-references to external databases Reaction and direction RHEA:15133 (master reaction) L-glutamate+H2O+NAD+ <?> 2-oxoglutarate+H++NADH+NH+4 RHEA:15134 - Left-to-Right (RHEA:15133, LR) L-glutamate+H2O+NAD+ ! 2-oxoglutarate+H++NADH+NH+4 RHEA:15135 - Left-to-Right (RHEA:15133, LR) 2-oxoglutarate+H++NADH+NH+4 ! L-glutamate+H2O+NAD+ RHEA:15136 - Bidirectional (RHEA:15133, BI) L-glutamate+H2O+NAD+ , 2-oxoglutarate+H++NADH+NH+4 and export, as well as validation and balancing. Rhea re- actions (like their constituent ChEBI entities) are manually curated and linked to a host of other resources, including underlying structural information. Each possible reaction in Rhea is represented by a unique ‘master reaction’ that is independent of any bio- logical context, having no associated directional informa- tion. Each such master reaction has the following attributes: . A unique identiﬁer . Two reaction parts (left and right) . A set of qualiﬁers that describe the type of reaction and if it is balanced . A curation status (approved, preliminary or obsolete) Each of the two reaction parts (arbitrarily deﬁned as left and right) are composed of a set of participant compounds, their stoichiometric coefﬁcient and possibly their localiza- tion. The compounds are deﬁned by a ChEBI identiﬁer, a name, a chemical formula, a net charge and possibly a 2D structure. The coefﬁcient may be an integer or a symbolic expression (n, n+1, n 1, etc). For transport reactions, nominal cellular compartments (localization) are speciﬁed by the tokens (in) and (out) using the side convention of NC-IUBMB. Each master reaction is uniquely represented at an ar- bitrarily chosen pH of 7.3. Rhea uses the Marvin pKa calculator from ChemAxon (http://www.chemaxon.com) to select the major species of each compound found at pH 7.3 and at a temperature of 298 K (9). For some com- pounds, such as phosphate, the major species at pH 7.3 (which is HPO42 ) may be present in only a slight excess over other minor species (such as H2PO4 ), so the reaction representation is a simpliﬁcation of the actual state. However, the selection of the major species serves to guar- antee data consistency, since the same protonation state will be used in all reactions (and the reactions are fully balanced, including protons). This convention is in accord with that adopted by some existing resources, such as Cross-references None UniPathway:UER00591 Reactome:REACT_710.4 Reactome:REACT_1896.4 UniPathway:UCR00243 KEGG:R00243 MetaCyc: GLUTAMATE-DEHYDROGENASE-RXN IntEnz: EC 1.4.1.2 IntEnz:EC 1.4.1.3 UniProt: DHE2_PORG3 UniProt: DHEA_NICPL,. . . MetaCyc (10), but contrasts with that adopted by others, such as KEGG (11) and BRENDA (12), which generally choose a neutral form for compound represen- tation. This latter convention has the advantage of being simple but prevents the balancing of reactions for charge and hydrogen atoms. Note that the export formats provided by Rhea allow users to compute structures at other pH values using commonly available chemo- informatics tools. To ensure data consistency, the following additional constraints are supported by the Rhea infrastructure: . The same compound (ChEBI ID, localization) cannot occur on both sides of a reaction. This results in the exclusion of certain common species, such as Mg2+ in Mg2+-ATP. . The reaction must be chemically balanced for mass and charge, i.e. the compounds found in the left and right parts of the equation must have the same total number of atoms of each type and the same net charge. . The reaction must be unique. To ensure this unique- ness, a ﬁngerprint is computed based on the com- pounds on both reaction parts (ChEBI ID, coefﬁcient, localization). Each unique master reaction is associated with three directional reactions—forward, reverse and bidirectional (reversible)—each of which has a distinct identiﬁer (Table 1). (Note that the validation steps described above for the master reaction are automatically applied to each of these directional reactions.) Rhea is therefore redundant, in the sense that each reaction has four distinct (validated) representations, but this feature allows direc- tional reactions from external resources to be linked to the appropriate directional reaction in Rhea (Table 1). The drawback of this approach is that Rhea may include dir- ectional reactions that are unfeasible in biological systems according to current knowledge.