The Structural Basis of Enzyme Halophilicity
Deborah G MADDOCKS*, David W HOUGH, and Michael J DANSON
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom
Haloferax volcanii is an extremely halophilic Archaeon that was originally isolated from hypersaline environments where the salt concentration approaches saturation (> 5 M NaC1). In order to survive at such extreme conditions the organism maintains an intracellular concentration of salt, mainly KC1, that is isotonic with its environment. Therefore, enzymes from extreme halophiles such as Hf. volcanii are fully active at salt concentrations up to 5 M and indeed they require these conditions in order to retain full activity. Thus enzymes from these organisms have potential biotechnological applications in both high salt environments and other situations of low water activity.
We have used citrate synthase as a model enzymefor an investigation of the structural basis of enzyme halophilicity. The gene encoding citrate synthase from Hf. volcanii has been cloned and sequenced. A structural model of the halophilic enzyme has been produced based on the crystal structure of citrate synthase from the hyperthermophilic Archaeon Pyro coccus furiosus. The model exhibits features typical of halophilic proteins, including a high density of negatively charged amino acids covering the surface of the protein. The contribution of the number and spatial arrangement of these residues to the halophilicity of the protein will be explored through site-directed mutagenesis and biophysical studies.
Investigation into the expression of the enzyme in its native host Haloferax is underway in conjunction with the heterologous expression of the enzyme in the non- halophilic bacterium E. coli. It is hoped to produce enough of the overexpressed protein from both host systems to undergo crystallisation trials. Crystallisation conditions will be based on the few crystallized halophilic proteins available. The crystal structure of citrate synthase will be compared with our model-based hypothesis, thus giving further insights into protein halophilicity.
