We have recently developed a new method for designing thermostable proteins using phylogenetic trees of enzymes. In this study, we investigated a method for designing proteins with improved stability using 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus as a model enzyme. We designed 12 mutant enzymes, each having an ancestral amino acid residue that was present in the common ancestor of Bacteria and Archaea. At least six of the 12 ancestral mutants tested showed thermal stability higher than that of the original enzyme. The results supported the hyperthermophilic universal ancestor hypothesis. The effect of ancestral residues on IPMDHs of several organisms and on the related enzyme isocitrate dehydrogenase was summarised and analysed. The effect of an ancestral residue on thermostability did not depend on the degree of conservation of the residue at the site, suggesting that the stabilisation of these mutant proteins is not related to sequence conservation but to the antiquity of the introduced residues. The results suggest also that this method could be an efficient way of designing mutant enzymes with higher thermostability based only on the primary structure and a phylogenetic tree. Study holds ProTherm entries: 18894, 18895, 18896, 18897, 18898, 18899, 18900, 18901, 18902, 18903, 18904, 18905, 18906, 18907, 18908, 18909, 18910, 18911, 18912, 18913, 18914, 18915, 18916, 18917, 18918, 18919 Extra Details: 0.5 mM EDTA was added in the experiment. The enzyme solution was incubated at various temperatures for 10 min,and cooled on ice. The amount of remained activity was estimated at pH 8.5, and 50 C. Tm is the apparent half-denaturation temperature T1/2. ancestral residue; protein stability; 3-isopropylmalate dehydrogenase; Thermus thermophilus; Commonote
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:51 p.m.
|Number of data points||50|
|Proteins||3-isopropylmalate dehydrogenase ; 3-isopropylmalate dehydrogenase|
|Assays/Quantities/Protocols||Experimental Assay: Tm details:Additives , prot_conc:5.4 microM, buffers:phosphate: 20 mM ; Experimental Assay: Tm prot_conc:10.9 microM, buffers:potassium phosphate: 20 mM, details:Additives EDTA (0.5 mM), ; Derived Quantity: dTm details:Additives , prot_conc:5.4 microM, buffers:phosphate: 20 mM ; Derived Quantity: dTm prot_conc:10.9 microM, buffers:potassium phosphate: 20 mM, details:Additives EDTA (0.5 mM),|
|Libraries||Mutations for sequence MKVAVLPGDGIGPEVTEAALKVLRALDEAEGLGLAYEVFPFGGAAIDAFGEPFPEPTRKGVEEAEAVLLGSVGGPKWDGLPRKIRPETGLLSLRKSQDLFANLRPAKVFPGLERLSPLKEEIARGVDVLIVRELTGGIYFGEPRGMSEAEAWNTERYSKPEVERVARVAFEAARKRRKHVVSVDKANVLEVGEFWRKTVEEVGRGYPDVALEHQYVDAMAMHLVRSPARFDVVVTGNIFGDILSDLASVLPGSLGLLPSASLGRGTPVFEPVHGSAPDIAGKGIANPTAAILSAAMMLEHAFGLVELARKVEDAVAKALLETPPPDLGGSAGTEAFTATVLRHLA|