Homology Modeling and Analysis of Vacuolar Aspartyl Protease from a Novel Yeast Expression Host Meyerozyma guilliermondii Strain SO

In eukaryotes, aspartyl protease (PEP4) is a localized hydrolase. PEP4 was recently identified from Meyerozyma guilliermondii strain SO (MgPEP4), a novel yeast expression host. But little is known about the structural properties and its catalytic mechanism. Multiple sequence alignment with other yeast aspartyl proteases revealed the conserved regions of MgPEP4 which belongs to the pepsin/proteinase_A_fungi superfamily. Two catalytic aspartic acid residues (Asp112 and Asp297) existed as a single copy at the DTG motif, a pattern of short conserved amino acids sequence. Homology modeling of MgPEP4 was done using Saccharomyces cerevisiae PEP4 (PDB ID: 1DPJ) as template. Using in silico analysis, we aim to reveal its stability by way of disulfide bridge formation and the catalytic mechanism of MgPEP4 with a universal protease inhibitor (pepstatin A). Structurally, only two out of the four conserved cysteine residues of the polypeptide were involved in intramolecular disulfide bridges in the validated structure as opposed to two disulfide bridges present in the template which conferred a critical stabilizing role in the protein structures. Pepstatin A (pepA) was docked at the substrate-binding site and showed hydrophilic interactions with the essential catalytic aspartic residues, which preliminarily proved the catalytic mechanism of MgPEP4. In conclusion, understanding of the structure and catalytic mechanism of MgPEP4 at the molecular level have given an insight about its role in the degradation of recombinant proteins in M. guilliermondii strain SO as an expression host as well as its potential applications in food, beverages, baking, leather and pharmaceutical industries. Further development of a new yeast strain could be done using MgPEP4 as the target protein.