Electrostatic interactions across the dimer-dimer interface contribute to the pH-dependent stability of a tetrameric malate dehydrogenase.
Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic bacteria usually are dimers. Using site-directed mutagenesis, we show here that a network of electrostatic interactions across the extra dimer-dimer interface in CaMDH is important for thermal stability and oligomeric integrity. Stability effects of single point mutations (E25Q, E25K, D56N, D56K) varied from -1.2 degrees C to -26.8 degrees C, and depended strongly on pH. Gel-filtration experiments indicated that the 26.8 degrees C loss in stability observed for the D56K mutant at low pH was accompanied by a shift towards a lower oligomerization state.
Department of Biology, University of Oslo, Box 1031 Blindern, N-0316 Oslo, Norway. alexandra.bjork@bio.uio.no
Alexandra
A
Bjørk
Dimitrios
D
Mantzilas
Reidun
R
Sirevåg
Vincent G H
VG
Eijsink
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry*; D019414:Chlorobi / Q000201:enzymology; D002850:Chromatography, Gel; D019281:Dimerization; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008291:Malate Dehydrogenase / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry; D055672:Static Electricity; D013696:Temperature
D001426:Bacterial Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D008291:Malate Dehydrogenase