Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface.


Abstract

We have investigated factors affecting stability at the subunit-subunit interface of the dimeric enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Bacillus subtilis. Site-directed mutagenesis was used to replace methionine 256, a key residue in the subunit interaction, with other amino acids. Thermal stability against irreversible inactivation of the mutated enzymes was examined by analyzing the residual activity after heat treatment. The mutations M256V and M256A increased thermostability by 2.0 and 6.0 degrees C, respectively, whereas the mutations M256L and M256I had no effect. Thermostability of the M256F mutated enzyme was 4.0 degrees C lower than that of the wild-type enzyme. To our surprise, increasing the hydrophobicity of residue 256 within the hydrophobic core of the enzyme resulted in a lower thermal stability. The mutated enzymes showed an inverse correlation between thermostability and the volume of the side chain at position 256. Based on the X-ray crystallographic structure of Escherichia coli IPMDH, the environment around M256 in the B.subtilis homolog is predicted to be sterically crowded. These results suggest that Met256 prevents favorable packing. Introduction of a smaller amino acid at position 256 improves the packing and stabilizes the dimeric structure of IPMDH. The van der Waals volume of the amino acid residue at the hydrophobic subunit interface is an important factor for maintaining the stability of the subunit-subunit interface and is not always optimized in the mesophilic IPMDH enzyme. Study holds ProTherm entries: 16587, 16588, 16589, 16590, 16591 Extra Details: dimeric enzyme; hydrophobic core; isopropylmalate dehydrogenase; subunit interaction; thermostability

Submission Details

ID: YpMCn3qQ3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:48 p.m.

Version: 1

Publication Details
Ohkuri T;Yamagishi A,Protein Eng. (2003) Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface. PMID:12968079
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 P05645 LEU3_BACSU