Studies on the Molecular Docking and Amino Acid Residues Involving in Recognition of Substrate in Proline Iminopeptidase by Site-Directed Mutagenesis

Abstract

The proline iminopeptidase (PchPiPA) of Phanerochaete chrysosporium catalyze specifically hydrolysis of N-terminal proline from peptides. The substrate Pro-pNA was docked into the catalytic pocket and several amino acid residues were identified to interact or associate with the substrate. Eight residues were selected for site-directed mutagenesis. The wild-type and mutant proteins were expressed in Escherichia coli and purified. Kinetic parameters were calculated by hydrolyzing Pro-pNA for these enzymes. Substitution of two Glu residues (Glu198 and Glu227) which interact with the substrate via formation of hydrogen bond, led to deleterious effect on catalytic efficiency (k(cat)/K(m)) due to decrease of k(cat) and increase of K(m). Four Phe residues consisting of catalytic pocket and surrounding the docked substrate, were substituted with Ala, resulting in decrease in k(cat)/K(m) to various extents. Substitution of two residues (Val267 and Cys267) localized at the deep end of the catalytic pocket also yielded negative influence on the substrate hydrolysis. Besides, all the mutants except E227Q exhibited lower thermostability than the wild-type did, indicating that these mutations may modulate the local structure. In conclusion, these amino acid residues may play an important role in maintaining local environment of the impacted catalytic pocket and be involved in recognizing or positioning the substrate.

Comparative Proteomics Reveals the Difference in Root Cold Resistance between Vitis. riparia × V. labrusca and Cabernet Sauvignon in Response to Freezing Temperature

“…The ABHD family of proteins in plants influences the lipid biosynthesis more towards leaf lipids, such as galactolipids, and less towards storage lipids [ 45 ]. The ProIP specifically catalyzes hydrolysis of N-terminal proline from peptides [ 46 ]. The MT is involved in the sterol and steroid biosynthesis [ 47 ].…”

Molecular Docking Analysis of Ficus religiosa Active Compound with Anti-Inflammatory Activity by Targeting Tumour Necrosis Factor Alpha and Vascular Endothelial Growth Factor Receptor in Diabetic Wound Healing

“…It must be separated and stored in a.pdb file format before simulating the molecular interactions. We used native ligand inhibitors provided on the PDB website for comparison with compound ligands from F. religiosa [18], [19].…”

Molecular Docking Approach of Natural Compound from Herbal Medicine in Java against Severe Acute Respiratory Syndrome Coronavirus-2 Receptor

“…Molecular docking has shown great promise as a new tool for discovering novel small molecule drugs with high protein targeting potential. In silico drug design interactions can predict the ligand-target protein interaction mechanism and the bond energy that occurs [22], [23]. Based on the concepts above, this study was conducted to identify the potential herbal medicines through in silico approach as the candidate for treating the transmission of COVID-19.…”