Cold-adaptation mechanism of mutant enzymes of 3-isopropylmalate dehydrogenase from Thermus thermophilus.


Abstract

Random mutagenesis of Thermus thermophilus 3-isopropylmalate dehydrogenase revealed that a substitution of Val126Met in a hinge region caused a marked increase in specific activity, particularly at low temperatures, although the site is far from the binding residues for 3-isopropylmalate and NAD. To understand the molecular mechanism, residue 126 was substituted with one of eight other residues, Gly, Ala, Ser, Thr, Glu, Leu, Ile or Phe. Circular dichroism analyses revealed a decreased thermal stability of the mutants (Delta T ((1/2))= 0-13 degrees C), indicating structural perturbations caused by steric conflict with surrounding residues having larger side chains. Kinetic parameters, k(cat) and K(m) values for isopropylmalate and NAD, were also affected by the mutation, but the resulting k(cat)/K(m) values were similar to that of the wild-type enzyme, suggesting that the change in the catalytic property is caused by the change in free-energy level of the Michaelis complex state relative to that of the initial state. The kinetic parameters and activation enthalpy change (Delta H (double dagger)) showed good correlation with the van der Waals volume of residue 126. These results suggested that the artificial cold adaptation (enhancement of k(cat) value at low temperatures) resulted from the destabilization of the ternary complex caused by the increase in the volume of the residue at position 126. Study holds ProTherm entries: 15168, 15169, 15170, 15171, 15172, 15173, 15174, 15175, 15176 Extra Details: cold adaptation; 3-isopropylmalate dehydrogenase; mutagenesis; thermophile enzyme

Submission Details

ID: dhu8NWVP

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Suzuki T;Yasugi M;Arisaka F;Oshima T;Yamagishi A,Protein Eng. (2002) Cold-adaptation mechanism of mutant enzymes of 3-isopropylmalate dehydrogenase from Thermus thermophilus. PMID:12082165
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
1WAL 1999-05-17T00:00:00+0000 2.27 3-ISOPROPYLMALATE DEHYDROGENASE (IPMDH) MUTANT (M219A)FROM THERMUS THERMOPHILUS
1DPZ 1999-12-29T00:00:00+0000 2.8 STRUCTURE OF MODIFIED 3-ISOPROPYLMALATE DEHYDROGENASE AT THE C-TERMINUS, HD711
1DR0 2000-01-06T00:00:00+0000 2.2 STRUCTURE OF MODIFIED 3-ISOPROPYLMALATE DEHYDROGENASE AT THE C-TERMINUS, HD708
1DR8 2000-01-06T00:00:00+0000 2.7 STRUCTURE OF MODIFIED 3-ISOPROPYLMALATE DEHYDROGENASE AT THE C-TERMINUS, HD177
1G2U 2000-10-21T00:00:00+0000 2.1 THE STRUCTURE OF THE MUTANT, A172V, OF 3-ISOPROPYLMALATE DEHYDROGENASE FROM THERMUS THERMOPHILUS HB8 : ITS THERMOSTABILITY AND STRUCTURE.
1GC8 2000-07-27T00:00:00+0000 2.5 THE CRYSTAL STRUCTURE OF THERMUS THERMOPHILUS 3-ISOPROPYLMALATE DEHYDROGENASE MUTATED AT 172TH FROM ALA TO PHE
1GC9 2000-07-28T00:00:00+0000 2.3 THE CRYSTAL STRUCTURE OF THERMUS THERMOPHILUS 3-ISOPROPYLMALATE DEHYDROGENASE MUTATED AT 172TH FROM ALA TO GLY
1HEX 1994-09-09T00:00:00+0000 2.5 STRUCTURE OF 3-ISOPROPYLMALATE DEHYDROGENASE IN COMPLEX WITH NAD+: LIGAND-INDUCED LOOP-CLOSING AND MECHANISM FOR COFACTOR SPECIFICITY
1IDM 1995-05-19T00:00:00+0000 2.2 3-ISOPROPYLMALATE DEHYDROGENASE, LOOP-DELETED CHIMERA
1IPD 1992-01-29T00:00:00+0000 2.2 THREE-DIMENSIONAL STRUCTURE OF A HIGHLY THERMOSTABLE ENZYME, 3-ISOPROPYLMALATE DEHYDROGENASE OF THERMUS THERMOPHILUS AT 2.2 ANGSTROMS RESOLUTION

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