The accumulation of sequencing data at a vertiginous rate necessitates annotation of the functions by automated methods without human expert input. The function predicting algorithms, while continually improving, do not enable reliable prediction of the functions of genes. The algorithms are nonetheless sufficiently effective to orient experimental work. In this context, we have deployed a strategy for the discovery of multiple enzyme activities catalyzed in families of proteins of unknown function or with a small number of associated functions. The approach is based on definition of a generic reaction conserved in the family, high-throughput enzymatic screening of representatives, structural investigations and genomic and metabolic context analysis. We applied the strategy to a Pfam family of unknown function and discovered 14 new enzymatic activities. In addition, we have proposed a metabolic role for some of those enzymes and found key residues liable to orient functional annotation. The extension of the strategy to other families of enzymes satisfying a few prerequisites will enable further investigation of the hidden functional diversity of enzymes (Bastard K. et al., Nat chem biol 2014).

Stylized 3-dimensional structure of a Pfam family member studied.