Classification of the caspase–hemoglobinase fold: Detection of new families and implications for the origin of the eukaryotic separins

Abstract A comprehensive sequence and structural comparative analysis of the caspase–hemoglobinase protein fold resulted in the delineation of the minimal structural core of the protease domain and the identification of numerous, previously undetected members, including a new protease family typified by the HetF protein from the cyanobacterium Nostoc . The first bacterial homologs of legumains and hemoglobinases were also identified. Most proteins containing this fold are known or predicted to be active proteases, but multiple, independent inactivations were noticed in nearly all lineages. Together with the tendency of caspase‐related proteases to form intramolecular or intermolecular dimers, this suggests a widespread regulatory role for the inactive forms. A classification of the caspase–hemoglobinase fold was developed to reflect the inferred evolutionary relationships between the constituent protein families. Proteins containing this domain were so far detected almost exclusively in bacteria and eukaryotes. This analysis indicates that caspase–hemoglobinase‐fold proteases and their inactivated derivatives are widespread in diverse bacteria, particularly those with a complex development, such as Streptomyces , Anabaena , Mesorhizobium , and Myxococcus . The eukaryotic separin family was shown to be most closely related to the mainly prokaryotic HetF family. The phyletic patterns and evolutionary relationships between these proteins suggest that they probably were acquired by eukaryotes from bacteria during the primary, promitochondrial endosymbiosis. A similar scenario, supported by phylogenetic analysis, seems to apply to metacaspases and paracaspases, with the latter, perhaps, being acquired in an independent horizontal transfer to the eukaryotes. The acquisition of the caspase–hemoglobinase‐fold domains by eukaryotes might have been critical in the evolution of important eukaryotic processes, such as mitosis and programmed cell death. Proteins 2002;46:355–367. © 2002 Wiley‐Liss, Inc.