PDBsum entry 1uxh

Oxidoreductase

PDB id: 1uxh

Contents

Protein chains

308 a.a. *

Ligands

NAD ×2

FUM ×2

Waters ×453

* Residue conservation analysis

References listed in PDB file

Key reference

Title

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

Authors

A.Bjørk, B.Dalhus, D.Mantzilas, R.Sirevåg, V.G.Eijsink.

Ref.

J Mol Biol, 2004, 341, 1215-1226. [DOI no: 10.1016/j.jmb.2004.06.079]

PubMed id

15321717

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.

Figure 1.
Figure 1. The structure of homotetrameric MDH from C. aurantiacus is shown in a ribbon diagram in the upper panel. The tetramer consists of two dimers (AB and CD). The NAD cofactor bound to the catalytic centre is shown in yellow. The rectangle indicates the location of the electrostatic network around Glu165 at the dimer--dimer interface in CaMDH (note that the network occurs four times in the tetramer). The lower panel shows a stereo view of the environment of Glu165 in subunit A, with backbones coloured according to the scheme used in the upper panel. The side-chains are shown in ball and stick in atom colours, Cd 2C bound to Glu165 is shown in yellow. Dotted lines indicate interacting atoms, located within 3 Å of each other.

Figure 5.
Figure 5. Stereo pictures showing the environment of residue 165 in cadmium-containing structures of wild-type CaMDH (A) and the E165Q (B) and E165K (C) mutants. (B) and (C) show 2F oKFc maps contoured at 1s for residue 165 to illustrate the conformation of the side-chains as well as the general quality of the refinement. Red spheres represent water molecules; the yellow sphere represents cadmium. In CaMDH (A) the negatively charged Glu165 binds directly to one of the cadmium ions in the structure (closest distance 2.5 A š ). In the E165Q mutant (B) the cadmium ion binds to residue 165 via a water molecule (closest Cd 2C -165 distance 4.2 A š ). In the E165K mutant (A) the cadmium ion has moved even further away from its original position and is now very close (2.3 A š , as compared to 4.2 A š in the wild-type) to Glu238 of the other monomer. The distance between cadmium and the z-amino group of Lys165 is 4.1 A š . Figure 1 and this Figure were made using the programs Bobscript 64 and Raster 3D. 65

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 341, 1215-1226) copyright 2004.