Ryder Stephanie, Pedigo Jacob, Ojennus Deanna Dahlke
Department of Chemistry, Whitworth University, 300 W. Hawthorne Rd., Spokane, Washington 99251, United States.
ACS Omega. 2023 Sep 14;8(38):35410-35416. doi: 10.1021/acsomega.3c05639. eCollection 2023 Sep 26.
Prolyl aminodipeptidase (PepX) is an α/β hydrolase that cleaves at penultimate N-terminal prolyl peptide bonds. The crystal structure of PepX from 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.
脯氨酰氨基二肽酶（PepX）是一种α/β水解酶，可在倒数第二个N端脯氨酰肽键处进行切割。来自[具体来源未提及]的PepX晶体结构在催化结构域内呈现出一个钙结合环。该环内的xDxDxDGxxD钙结合序列在乳酸菌的PepX蛋白中高度保守，但其作用仍不清楚。在金属螯合剂乙二胺四乙酸（EDTA）存在的情况下，以及在过量钙离子存在的情况下，酶活性均未受到显著影响。为了消除钙结合，在保守的钙结合序列基序内构建了D196A和D194A/D196A突变体。通过比色动力学分析和蛋白质热迁移分析，D196A突变体的酶活性和稳定性与野生型酶相当。然而，D194A/D196A突变体虽然保留了类似天然的结构和热稳定性，但却没有活性，这与EDTA和钙滴定结果相矛盾。这表明钙与PepX的结合可能对活性至关重要。
