Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface.


Abstract

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability. Study holds ProTherm entries: 17252, 17253, 17254, 17255, 17256, 17257, 17258, 17259, 17260 Extra Details: tetrameric malate dehydrogenase; thermal stability; site-directed mutagenesis; electrostatic subunit interactions; crystal structures

Submission Details

ID: 3UEGBsu44

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:49 p.m.

Version: 1

Publication Details
Bjørk A;Dalhus B;Mantzilas D;Sirevåg R;Eijsink VG,J. Mol. Biol. (2004) Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface. PMID:15321717
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