Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface.


Abstract

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic organisms usually are dimers. To investigate the potential contribution of the extra dimer-dimer interface in CaMDH with respect to thermal stability, we have engineered an intersubunit disulfide bridge designed to strengthen dimer-dimer interactions. The resulting mutant (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a 200-fold increase in half-life at 75 degrees C and an increase of 15 deg. C in apparent melting temperature compared to the wild-type. The crystal structure of the mutant (solved at 1.75 A resolution) was essentially identical with that of the wild-type, with the exception of the added inter-dimer disulfide bridge and the loss of an aromatic intra-dimer contact. Remarkably, the mutant and the wild-type had similar temperature optima and activities at their temperature optima, thus providing a clear case of uncoupling of thermal stability and thermoactivity. The results show that tetramerization may contribute to MDH stability to an extent that depends strongly on the number of stabilizing interactions in the dimer-dimer interface. Study holds ProTherm entries: 16787, 16788 Extra Details: 5 mM DTT was added in the experiment. tetrameric malate dehydrogenase; thermal stability; mutagenesis; disulfide bridge; crystal structure

Submission Details

ID: 6htN7Qzb3

Submitter: Connie Wang

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

Version: 1

Publication Details
Bjørk A;Dalhus B;Mantzilas D;Eijsink VG;Sirevåg R,J. Mol. Biol. (2003) Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface. PMID:14636605
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
4CL3 2014-02-05 1.7 1.70 A resolution structure of the malate dehydrogenase from Chloroflexus aurantiacus
1UR5 2003-11-05 1.75 Stabilization of a Tetrameric Malate Dehydrogenase by Introduction of a Disulfide Bridge at the Dimer/Dimer Interface
1UXJ 2004-08-26 1.75 Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface
1UXK 2004-08-26 1.8 Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface
1UXG 2004-08-26 1.9 Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface.
1UXI 2004-08-26 2.1 Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface
1UXH 2004-08-26 2.1 Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface
1GUY 2002-02-15 2.2 Structural Basis for Thermophilic Protein Stability: Structures of Thermophilic and Mesophilic Malate Dehydrogenases

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.5 Malate dehydrogenase B8GDA2 MDH_CHLAD
100.0 Malate dehydrogenase B9LLP6 MDH_CHLSY
100.0 Malate dehydrogenase P80040 MDH_CHLAA