DepoCatalog: mapping diversity of 129 recombinantly produced Klebsiella phage depolymerases

Our understanding of how depolymerase sequence and structure determine substrate specificity is fragmentary due to the limited number of experimentally characterized enzymes. Here we show DepoCatalog - an experimentally validated collection of 129 recombinantly prepared Klebsiella phage depolymerases (90 enzymes produced in this study and 39 homologs from the literature), with specificity spanning 75 KL-types. Enzymes originated from podo-, sipho-, myo-, jumbo phages, and prophages. Using activity profiling, structural modeling, and domain dissection, we propose a five‑class framework that captures the architectural and functional diversity of these enzymes. DepoCatalog uncovers cross-reactivity and taxa‑specific enzymes. Structural comparisons indicate that specificity switching or extension is associated with modifications to the C‑terminal domain. We further hypothesize that podoviruses encoding up to two RBPs show greater receptor adaptability than jumbo phages with multiple specialized RBPs. Finally, we develop a publicly accessible, DepoCat dataset (https://depocat.uwr.edu.pl) for structural classification and comparison of newly identified depolymerases.