Enzymatic and structural studies of peruvianin-I: the first germin-like protein with proteolytic activity

Abstract

The germin-like protein (GLP) purified from Thevetia peruviana, Peruvianin-I, is the only one described as having proteolytic activity. Therefore, the goal of this study was to investigate the structural features responsible for its enzymatic activity. Although the amino acid sequence of Peruvianin-I showed high identity with other GLPs, it exhibited punctual mutations, which were responsible for the absence of oxalate oxidase activity. The phylogenetic analysis showed that Peruvianin-I does not belong to any classification of GLP subfamilies. Moreover, Peruvianin-I contains a catalytic triad found in all plant cysteine peptidases. Molecular docking simulations confirmed the role of the catalytic triad in its proteolytic activity. Synchrotron radiation circular dichroism assays confirmed that Peruvianin-I was stable at pH ranging from 5.0 to 8.0 and that it presented significant structural changes only above 60 °C. The addition of iodoacetamide caused changes in its native conformation, but only a slight effect was observed after adding a reducing agent. The x-ray crystal structure of Peruvianin-I was solved to a nominal resolution of 2.15 Å. The overall structure of peruvianin-I shows an arrangement composed by monomers locked into a homohexamer (a trimer of dimers). The peruvianin-I monomers adopts the typical β-barrel fold of the Germin-like proteins (GLPs). In contrast to Germins and GLPs, Peruvian-I does not present ions inside the monomers. Our results showed through of electron density map the presence of two glycan-binding sites located at ASN55 and ASN144. This study reports an unusual protein with germin-like structure, lacking typical oxalate oxidase activity. Instead, the proteolytic activity observed suggests that the protein is a cysteine peptidase. Details of the active site were used to present and discuss a plausible mechanism of action for Peruvianin-I. These structural peculiarities make Peruvianin-I an interesting model for further understanding of the action of laticifer fluids in plant defense.