Domain-based and family-specific sequence identity thresholds increase the levels of reliable protein function transfer.

Divergence in function of homologous proteins is based on both sequence and structural changes. Overall enzyme function has been reported to diverge earlier (50% sequence identity) than overall structure (35%). We herein study the functional conservation of enzymes and non-enzyme sequences using the protein domain families in CATH-Gene3D. Despite the rapid increase in sequence data since the last comprehensive study by Tian and Skolnick, our findings suggest that generic thresholds of 40% and 60% aligned sequence identity are still sufficient to safely inherit third-level and full Enzyme Commission numbers, respectively. This increases to 50% and 70% on the domain level, unless the multi-domain architecture matches. Assignments from the Kyoto Encyclopedia of Genes and Genomes and the Munich Information Center for Protein Sequences Functional Catalogue seem to be less conserved with sequence, probably due to a more pathway-centric view: 80% domain sequence identity is required for safe function transfer. Comparing domains (more pairwise relationships) and the use of family-specific thresholds (varying evolutionary speeds) yields the highest coverage rates when transferring functions to model proteomes. An average twofold increase in enzyme annotations is seen for 523 proteomes in Gene3D. As simple 'rules of thumb', sequence identity thresholds do not require a bioinformatics background. We will provide and update this information with future releases of CATH-Gene3D.