Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus.


Abstract

A newly selected cold-adapted mutant 3-isopropylmalate dehydrogenase (IPMDH) from a random mutant library was a double mutant containing the mutations I11V and S92F that were found in cold-adapted mutant IPMDHs previously isolated. To elucidate the effect of each mutation on enzymatic activity, I11V and six multiple mutant IPMDHs were constructed and analyzed. All of the multiple mutant IPMDHs were found to be improved in catalytic activity at moderate temperatures by increasing the k(cat) with a simultaneous increase of K(m) for the coenzyme NAD(+). k(cat) was improved by a decrease in the activation enthalpy, DeltaH( not equal). The multiple mutants did not show large reduction in thermal stability, and one of them showed enhanced thermal stability. Mutation from I11 to V was revealed to have a stabilizing effect. Mutants showed increased thermal stability when the mutation I11V was combined. This indicates that it is possible to construct mutants with enhanced thermal stability by combining stabilizing mutation. No additivity was observed for the thermodynamic properties of catalytic reaction in the multiple mutant IPMDHs, implying that the structural changes induced by the mutations were interacting with each other. This indicates that careful and detailed tuning is required for enhancing activity in contrast to thermal stability. Study holds ProTherm entries: 12093, 12094, 12095, 12096, 12097, 12098, 12099, 12100, 12101, 12102, 12103 Extra Details: additive effect; cold adaptation; 3-isopropylmalate dehydrogenase;,kinetic analysis; thermophilic enzyme; thermal stability

Submission Details

ID: p927fDZQ

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:43 p.m.

Version: 1

Publication Details
Yasugi M;Suzuki T;Yamagishi A;Oshima T,Protein Eng. (2001) Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus. PMID:11579229
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