Abstract
Prolyl aminodipeptidase (PepX) is an α/β hydrolase that cleaves at penultimate N-terminal prolyl peptide bonds. The crystal structure of PepX from Lactobacillus helveticus exhibits a calcium-binding loop within the catalytic domain. The calcium-binding sequence of xDxDxDGxxD within this loop is highly conserved in PepX proteins among lactic acid bacteria, but its purpose remains unknown. Enzyme activity is not significantly affected in the presence of the metal chelator ethylenediaminetetraacetic acid (EDTA), nor in the presence of excess calcium ions. To eliminate calcium binding, D196A and D194A/D196A mutations were constructed within the conserved calcium-binding sequence motif. Enzyme activity and stability of the D196A mutant were comparable to the wild-type enzyme by colorimetric kinetic assays and protein thermal shift assays. However, the D194A/D196A mutant was inactive though it retained native-like structure and thermal stability, contradicting the EDTA and calcium titration results. This suggests calcium binding to PepX may be essential for activity.