Investigating the Thermostability of Mycoplasma Arginine Deiminase: a Protein Simulation Approach

Engineering the proteins to improve their thermostability is crucial for expanding their industrial and biotechnological applications. Targeted mutagenesis at flexible regions has been proved to be an efficient method to enhance the protein thermostability. Arginine deiminase (ADI), an Arg-catabolizing enzyme, is a potential antitumor agent for treating Arg-auxotrophic cancers, such as hepatocellular carcinoma and melanoma. It has also industrial potential application in measuring L-Arg concentration in beverages as well as the enzymatic production of L-Cit from L-Arg. In this study, molecular dynamics (MD) simulation was performed to investigate the stability and flexibility of the ADI from Mycoplasma arginini (MaADI) at two different temperatures (300 and 330 K), and subsequently to rigidify the most flexible regions through mutagenesis to enhance the enzyme stability. MaADI showed instability at 330 K. Loop L5 (102–106), and L23 (299–308) residues were predicted as the highest flexible regions, and, hence, were chosen as the mutation target. Eight variants were designed by mutating higher flexible residues of Loop23, N301-A304, based on consensus and/or most frequent naturally occurred residues for each position and ΔΔG. The stability of the modeled variant structures was investigated through MD simulation. G303D and A304G showed decreased flexibility of the residues and were introduced as more stable variants. As far as we know, no proper investigation has been performed on the MaADI stability before. Our results can provide a useful guide for engineering Mycoplasmal ADI to improve its properties.