Thousands of biochemical reactions with characterized activities are orphan, meaning they cannot be assigned to a specific enzyme, leaving gaps in metabolic pathways. Novel reactions predicted by pathway-generation tools also lack associated sequences, limiting protein engineering applications. Associating orphan and novel reactions with known biochemistry and suggesting enzymes to catalyze them is a daunting problem. We propose a new method, BridgIT, to identify candidate genes and protein

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... ences for these reactions, and this method introduces, for the first time, information about the enzyme binding pocket into reaction similarity comparisons. We performed two large-scale validation studies to test BridgIT predictions against experimental biochemical evidence. For the 234 orphan reactions from KEGG 2011 that became non-orphan in KEGG 2018, BridgIT predicted the exact or a highly related enzyme for 211 of them. Moreover, for 334 out of 379 novel reactions in 2014 that were later catalogued in KEGG 2018, BridgIT predicted the exact or highly similar enzyme sequences. BridgIT requires knowledge about only three connecting bonds around the atoms of the reactive sites to correctly identify protein sequences for 93% of analyzed enzymatic reactions.