Site-directed mutagenesis and halophilicity of dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii.
A homology-modelled structure of dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been generated using the crystal structure of the enzyme from Pseudomonas fluorescens. Analysis of the halophilic enzyme structure identified a potential K+-binding site comprising four co-ordinated glutamate residues (E423 and E426 from each monomer) at the subunit interface of the dimeric protein. Whilst E426 is conserved throughout non-halophilic dihydrolipoamide dehydrogenases, E423 is only present in the halophilic enzyme. Four site-directed mutations of the Haloferax dihydrolipoamide dehydrogenase have been made (E423D, E423Q, E423S, and E423A) and the recombinant mutants expressed and characterised. From an analysis of their kinetic properties, salt-dependent activities and thermal stabilities, it is concluded that this site has an important influence on the halophilicity of the enzyme. The findings support the view that the arrangement and interaction of the negatively charged amino acids are as important as the total net charge in determining the adaptation of proteins to high salt concentrations.
Jolley
K A
KA
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, UK.
Russell
R J
RJ
Hough
D W
DW
Danson
M J
MJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D019622:Haloferax volcanii / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011188:Potassium / Q000378:metabolism; D011189:Potassium Chloride / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D011189:Potassium Chloride; D008058:Dihydrolipoamide Dehydrogenase; D011188:Potassium