Harnessing natural diversity to identify key residues in Prolidase

Prolidase ( PEPD) catalyses the cleavage of dipeptides with high affinity for proline at the C-terminal end. This function is required in almost all living organisms and orthologues of PEPDwere thus detected across a broad taxonomic range. In order to detect strongly conservedresidues in PEPD, we analysed PEPDorthologous sequences identified in data sets of animals, plants, fungi, archaea, and bacteria. Due to conservationover very long evolutionary time, conservedresidues are likely to be of functional relevance. Single amino acid mutations in PEPDcause an autosomal disorder called prolidase deficiencyand were associated with various cancer types. We provide new insights into 15 additional residues with putative roles in prolidase deficiencyand cancer. Moreover, our results confirm previous reports identifying five residues involved in the binding of metal cofactors as highly conservedand enable the classification of several non-synonymous single nucleotide polymorphisms as likely pathogenic and seven as putative polymorphisms. Moreover, more than 50 conservedresidues across species, which were not previously described, were identified. Conservationdegree per residue across the animal kingdom were mapped to the human PEPD3D structure revealing the strongest conservationclose to the active site accompanied with a higher functional implication and pathogenic potential, validating the importance of a characteristic active site fold for prolidase identity.