Investigating the Effect of Targeted G207W Mutation to Reduce Al losteric Regulation in Aspartate Kinase III of Acinetobacter

Abstract Aspartate Kinase (AK) is an allosteric enzyme responsible for the committed step in the biosynthesis of the aspartate family amino acids, including threonine,lysine, methionine, and isoleucine. This critical enzyme is allosterically regulated by pathway end products. In most microorganisms, there are three isozymes responsible for phosphorylating aspartate, namely AKI, AKII, and AKIII. All three isozymes are allosterically inhibited by high concentrations of amino acids produced at the end of the pathway. Binding of free cytoplasmic lysine to AKIII allosterically diminishes the activity of this isozyme by converting the R conformation to the T conformation. Structure prediction of AKIII of Acinetobacter and comparison with the available structures of E. coli's AKIII revealed high similarities including evolutionarily conserved residues. We aimed to develop a novel protocol for identifying key amino acids in protein structures by integrating two complementary approaches: multiple sequence alignment (MSA), which highlights evolutionarily conserved residues under natural selection pressure, and residue interaction networks (RINs) analysis, which reveals structural roles in both active and inactive conformations of AKIII of Acinetobacter. Our results underscored the pivotal role of G207 in facilitating the conversion of the R state to the T state, leading to allosteric inhibition of the catalytic activity of this isozyme. Based on molecular dynamics simulation methods, we performed in-silico single mutations at positions that presumably hinder the R to T state conversion. Our results suggested that G207W point mutation is arguably the most effective manipulation keeping AKIII active even at high cytoplasmic concentrations of L-lysine.