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


Abstract

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 PDB Entries

Structure ID Release Date Resolution Structure Title
2Y3Z 2011-01-19 1.83 Structure of Isopropylmalate dehydrogenase from Thermus thermophilus - apo enzyme
4F7I 2012-06-13 2.0 Structure of Isopropylmalate dehydrogenase from Thermus thermophilus in complex with IPM, Mn and NADH
4WUO 2014-11-12 2.05 Structure of the E270A Mutant Isopropylmalate dehydrogenase from Thermus thermophilus in complex with IPM, Mn and NADH
1G2U 2000-11-01 2.1 THE STRUCTURE OF THE MUTANT, A172V, OF 3-ISOPROPYLMALATE DEHYDROGENASE FROM THERMUS THERMOPHILUS HB8 : ITS THERMOSTABILITY AND STRUCTURE.
1XAA 1996-04-03 2.1 3-ISOPROPYLMALATE DEHYDROGENASE, LOW TEMPERATURE (100K) STRUCTURE
1XAD 1996-04-03 2.1 CHIMERA ISOPROPYLMALATE DEHYDROGENASE BETWEEN BACILLUS SUBTILIS (M) AND THERMUS THERMOPHILUS (T) FROM N-TERMINAL: 20% T MIDDLE 20% M RESIDUAL 60% T, MUTATED AT S82R. LOW TEMPERATURE (150K) STRUCTURE.
1XAB 1996-04-03 2.1 3-ISOPROPYLMALATE DEHYDROGENASE, LOW TEMPERATURE (150K) STRUCTURE
1XAC 1996-04-03 2.1 CHIMERA ISOPROPYLMALATE DEHYDROGENASE BETWEEN BACILLUS SUBTILIS (M) AND THERMUS THERMOPHILUS (T) FROM N-TERMINAL: 20% T MIDDLE 20% M RESIDUAL 60% T, MUTATED AT S82R. LOW TEMPERATURE (100K) STRUCTURE.
1IDM 1995-09-15 2.2 3-ISOPROPYLMALATE DEHYDROGENASE, LOOP-DELETED CHIMERA
1DR0 2000-01-19 2.2 STRUCTURE OF MODIFIED 3-ISOPROPYLMALATE DEHYDROGENASE AT THE C-TERMINUS, HD708
1IPD 1993-10-31 2.2 THREE-DIMENSIONAL STRUCTURE OF A HIGHLY THERMOSTABLE ENZYME, 3-ISOPROPYLMALATE DEHYDROGENASE OF THERMUS THERMOPHILUS AT 2.2 ANGSTROMS RESOLUTION
2Y41 2011-01-19 2.2 Structure of Isopropylmalate dehydrogenase from Thermus thermophilus - complex with IPM and MN
1WAL 1999-05-25 2.27 3-ISOPROPYLMALATE DEHYDROGENASE (IPMDH) MUTANT (M219A)FROM THERMUS THERMOPHILUS
1GC9 2000-09-27 2.3 THE CRYSTAL STRUCTURE OF THERMUS THERMOPHILUS 3-ISOPROPYLMALATE DEHYDROGENASE MUTATED AT 172TH FROM ALA TO GLY
1OSJ 1997-01-27 2.35 STRUCTURE OF 3-ISOPROPYLMALATE DEHYDROGENASE
1HEX 1994-12-20 2.5 STRUCTURE OF 3-ISOPROPYLMALATE DEHYDROGENASE IN COMPLEX WITH NAD+: LIGAND-INDUCED LOOP-CLOSING AND MECHANISM FOR COFACTOR SPECIFICITY
2Y40 2011-01-19 2.5 Structure of Isopropylmalate dehydrogenase from Thermus thermophilus - complex with Mn
1GC8 2000-09-27 2.5 THE CRYSTAL STRUCTURE OF THERMUS THERMOPHILUS 3-ISOPROPYLMALATE DEHYDROGENASE MUTATED AT 172TH FROM ALA TO PHE
2Y42 2011-01-19 2.5 Structure of Isopropylmalate dehydrogenase from Thermus thermophilus - complex with NADH and Mn
1DR8 2000-01-19 2.7 STRUCTURE OF MODIFIED 3-ISOPROPYLMALATE DEHYDROGENASE AT THE C-TERMINUS, HD177
2ZTW 2009-10-13 2.79 Structure of 3-isopropylmalate dehydrogenase in complex with the inhibitor and NAD+
1DPZ 2000-01-12 2.8 STRUCTURE OF MODIFIED 3-ISOPROPYLMALATE DEHYDROGENASE AT THE C-TERMINUS, HD711
1OSI 1997-01-27 3.0 STRUCTURE OF 3-ISOPROPYLMALATE DEHYDROGENASE

Relevant UniProtKB Entries

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