Investigation of His-His interaction on the TMGS stability

Methylglyoxal synthase is a homohexameric protein that catalyzes an elimination reaction that converts dihydroxyacetone phosphate (DHAP) to form orthophosphate and methyglyoxal, which very applicable in industry. For this reason, MGS study and improved its stability is important. Recently, a gene encoding MGS from Thermus sp. GH5 (TMGS) was cloned, overexpressed and its structure has been studied by X-ray crystallography. These studies showed that TMGS gene was composed of 399 bp which encoded a polypeptide of 132 amino acids with a molecular mass of 14.3KDa. The crystal structure of TMGS revealed that His23, Arg22 and Phe19 are in closed distance and forming a loop. On the other hand our recent study has shown that His-His interaction may be important in protein stability. To further explore this phenomenon, in this survey two modified enzymes were produced with site directed mutagenesis (SDM); one of them, one histidine and another, two histidine inserted between Arg22 and His 23. After sequencing, expression and purification, thermal stability measurements performed and the data exhibited that the thermostabilities of both mutants improved compared native TMGS. These mutagenesis data supporte this hypothesis that the interactions between like- charged amino acid residues can improve protein stability.