Mutations of key substrate binding residues of leishmanial peptidase T alter its functional and structural dynamics

Abstract

BackgroundM20 aminopeptidases, such as Peptidase T (PepT), are implicated in the hydrolysis of oligopeptides during the terminal stages of protein degradation pathway to maintain turnover. Therefore, specific inhibition of PepT bores well for the development of novel next-generation antileishmanials. This work describes the metal dependence, substrate preferences and inhibition of PepT, and demonstrates in detail the role of its two conserved substrate binding residues. MethodsPepT was purified and characterized using a scheme of peptide substrates and peptidomimetic inhibitors. Residues T364 and N378 were mutated and characterized with an array of biochemical, biophysical and structural biology methods. ResultsPepT sequence carries conserved motifs typical of M20 peptidases and our work on its biochemistry shows that this cytosolic enzyme carries broad substrate specificity with best cleavage preference for peptides carrying alanine at the P1 position. Peptidomimetics amastatin and actinonin occupied S1 pocket by competing with the substrate for binding to active site and inhibited PepT potently, while arphamenine A and bestatin were less effective inhibitors. We further show that the mutation of conserved substrate binding residues (T364 and N378) to alanine affects structure, reduces substrate binding and alters the amidolytic activity of this dimeric enzyme. ConclusionsPepT preferentially hydrolyzes oligopeptides carrying alanine at P1 position and is potently inhibited by peptidomimetics. Reduced substrate binding after mutations was a key factor involved in amidolytic digressions. General significanceThis study provides insights for further exploration of the druggability of PepT and highlights prospective applications of this enzyme along with its mutazyme T364A/N378A.