ez-CAZy a reference annotation database for linking glycoside hydrolase sequence to enzymatic activity

Glycoside Hydrolases (GH) are carbohydrate-active enzymes that play a critical role in the degradation of carbohydrates, impacting ecosystem function, human health, and biotechnological applications. The functional annotation of GHs within the CAZy database is hindered by the lack of sequence-specific definitions, annotation tools, and reliance on generalized "majority rule" activity assumptions. Here, we introduce ez-CAZy, a custom reference database designed to link GH sequences, as well as other CAZy, to their enzymatic activities. By reannotating over 7,000 biochemically characterized GHs using Hidden Markov Model profiles and other publicly accessible tools, we provide detailed sequence metadata, domain architectures, and functional predictions. Our analysis reveals the clustered distribution of enzymatic activities and multi-domain architectures within GH families, facilitating more precise functional predictions for newly identified sequences. The predictive accuracy of ez-CAZy was validated using over 500 recently characterized GHs, demonstrating functional annotation. ez-CAZy addresses critical gaps in GH annotation pipelines and offers a publicly accessible tool to support sequence analysis and enzymatic research. This work underscores the need for standardized enzyme characterization and expanded substrate testing to enhance annotation accuracy in future studies.