Figure 2. The relationships between Rhea and databases of chemical compounds, proteins and enzyme classiﬁcation. ChEBI provides infor- mation on chemical compounds for the creation of chemical reactions in Rhea. The resulting Rhea reactions are used in IntEnz to describe the enzymes catalyzing the reactions. ENZYME is generated from IntEnz data and currently uses the textual representation reactions also used by the IUBMB EC list. Also shown are the cross-references provided by Rhea to other resources. These are generated manually and computationally. Rhea curation Each master reaction has a curation status, which is one of approved, preliminary or obsolete. To be approved, a reaction has to fulﬁll the constraints described in the previous section relating to unicity and balance. Reactions can also have descriptive qualiﬁers such as ‘chemically balanced’, ‘transport’, ‘class of reaction’ and ‘polymerization’. The manual curation process includes veriﬁcation of the selected ChEBI entities (assisted by chemoinformatic tools), the addition of original literature citations, and the addition of cross references to other resources. Rhea citations are managed in the EBI’s CiteXplore bibliog- raphy database (http://www.ebi.ac.uk/citexplore/). Rhea cross-references several resources describing biochemical reactions including KEGG (11), EcoCyc/MetaCyc (10,13) and UniPathway (14). As described above, these cross-references link speciﬁc directional instances of reac- tions in Rhea to those of other resources. Cross-references are also added automatically to Reactome (15) and MACiE (16) on the basis of their reaction participants (ChEBI compound) and the indicated reaction direction. An important goal of the Rhea project is to link the chemical information from ChEBI to that in resources describing enzymatic reactions, such as IntEnz (Integrated relational Enzyme database) (17) that contains data on enzymes organized by EC numbers (Figure 2). Rhea now provides the chemical representation for enzyme-catalyzed reactions in IntEnz (from which Rhea is cross-referenced). Rhea also includes cross- references to EC numbers, thus providing an entry point to IntEnz and enzyme classiﬁcation. EC numbers are subsequently used to propose cross-references to protein sequences in the UniProtKB/Swiss-Prot knowledgebase (18). Nucleic Acids Research, 2012, Vol. 40, Database issue D757 Rhea curation may also include the selection of descriptive names or labels for the participating ChEBI compounds, in order to provide a more easily understand- able (human readable) reaction description. It is import- ant to note that ChEBI and Rhea labels for a speciﬁc compound may differ. Rhea makes use of labels marked ‘UniProt synonym’ in ChEBI (these labels are so named as they will in future provide a controlled vocabulary for chemical compounds in UniProtKB). To illustrate this, CHEBI:15378 has the ChEBI common name hydron whereas Rhea uses the label H+. This practice of selecting easily understood chemical labels for Rhea means that the displayed label for certain chemical species will not neces- sarily show the correct charge state (a typical example being NAD+, CHEBI:57540, which is actually negatively charged). However, the underlying compounds are cor- rectly charged and the reaction is appropriately balanced. Comparison of Rhea and related resources When the Rhea project was initiated, the only freely avail- able database of reactions was the KEGG LIGAND database. During the intervening period, several addition- al resources containing information on chemical com- pounds and reactions have become available (while KEGG data now requires a subscription for download) (Table 2). Some of these newly available resources focus on enzymes catalyzing chemical reactions [e.g. BRENDA (12), ExplorEnz (2), Enzyme (3) and the aforementioned IntEnz (15)], while others provide detailed information on reaction mechanisms [e.g. EzCatDB (19), MACiE (16)] or kinetic data [e.g. BRENDA (12), SABIO-RK (20)]. Resources that provide comprehensive reaction data and are comparable in scope with Rhea include BioPath (21), KEGG (11) and MetaCyc (10). Submission to Rhea Rhea welcomes submissions describing reactions that are not currently available in Rhea. All new reaction submis- sions should be posted on our SourceForge Reaction Requests/Updates tracker (https://sourceforge.net/ projects/rhea-ebi/) with relevant information including ChEBI identiﬁers for each reaction participant and cross-references to other relevant databases and source literature where available. Rhea content At the time of writing, Rhea (release 24) includes 4321 master reactions (each associated with three directional reactions) that involve 3788 distinct ChEBI chemical entities. Among them, 251 are transport reactions. In the corresponding IntEnz database (release 71) 3145 of the 4596 enzyme entries (EC numbers) have their reactions described in Rhea. This corresponds to a total of 3658 distinct Rhea reactions (as there may be a many-to-many relationship between reactions and enzymes). The database is updated by monthly releases. Updates are synchronized with ChEBI releases.