Designing thermostable proteins: ancestral mutants of 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree.


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

Submission Details

ID: tTGCdptW

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Watanabe K;Ohkuri T;Yokobori S;Yamagishi A,J. Mol. Biol. (2006) Designing thermostable proteins: ancestral mutants of 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree. PMID:16309701
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 3-isopropylmalate dehydrogenase Q5SIY4 LEU3_THET8
99.4 3-isopropylmalate dehydrogenase P61494 LEU3_THET2
100.0 3-isopropylmalate dehydrogenase P61495 LEU3_THETH
91.9 3-isopropylmalate dehydrogenase P24098 LEU3_THEAQ