Several reactions in BRENDA could not be mapped by a single rule application. To address this, we developed an algorithm to allow for reactions occurring at multiple sites. For example, if a reaction yields the oxidation of two hydroxyl moieties in a molecule, the respective oxidation rule needs to be applied twice. We exhaustively enumerated the outcomes of applying a rule up to two times (the maximum number of steps can be customized in our software package). To speed up this enumeration, we used only the reaction rules already present in the EC class from step 4A (or the full set if no reaction rules were recorded yet). If multiple rule applications led to the desired outcome, the overall reaction was saved, as well as all the individual steps. As an example, Fig. depicts the oxidation of 1,2-butanediol, which requires rule application (oxidation by molecular oxygen) at two different sites. Since the order of the individual steps is not known, all four individual reaction steps are included in the database, and flagged with a keyword to indicate the reaction was obtained from a multi-step reaction. For individual reactions, molecules not participating were omitted, here for example the second oxygen molecule at step 1. If the obtained single-step reactions were the same due to symmetry, duplicates were dropped. Multi-step reactions as well as their corresponding single-step components were then corrected for stereoinformation similar to single step reactions.