Studies on the thermostabilities of the enzymes and the structures of the chimeric enzymes produced by the gene technology

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Chimeric 3-isopropylmalate dehydrogenase were expressed from the fused gene between the thermophilic and mesophilic bacteria. When we compared the thermostabililty of 4M6T, the 40% of N-terminus from mesophyll and the remaining 60% from thermophile, and 2T2M6T, the 20-40% of the N-terminal residues from mesophyll, the latter was more sensitive to the heat than the former, inspire of the less numbers of residues from thermophile. The other experiments were done to recover the thermostability of the enzyme through the replacement of a residue either by the site-directed mutagenesis or induced mutagenesis by heat. We found I93L-2T2M6T and S82R-2T2M6T as thermostable enzymes.With the present project, the structural studies of these molecules have been undertaken by the X-ray crystallography. The structures were refined within 2.1A^^ﾟ resolution by PROLSQ until the conventional R factors of 0.18-0.20. Each molecule was composed of two identical subunits each of which has 345 amino acid resi … More dues. The polypeptide chain of the subunit was folded into two domains, designated first(1-99 and 252-345) and second (100-251) domains, each of which has a topologically alpha/beta structure with the other. The long arm from 140 to 150 played roles for making a dimer by the hydrogen bonds between them. The structures of chimeric enzymes differs little from thermophile. The rms deviations of the Ca atoms were 0.3A^^ﾟ for 4M6T and 0.4A^^ﾟ for 2T2M6T, respectively. The large deviations common to the two chimeras were found the loop around the 80. This movements may have something to do with the thermostability. The mutant I93L-2T2M6T released the stress between the isoleucine residue and the peptide chain by replacing it to leucine. In case of S82R-2T2M6T, the extra hydrogen bond between replaced arginine and Glu89 via water molecule. This hydrogen bond may fix the chain folding of the loop, and improve its thermostability. Many factors like entropy and enthalpy have to be taken into consideration to understand the stability of the enzyme. Less