SITE-DIRECTED MUTAGENESIS AND HALOPHILICITY OF DIHYDROLIPOAMIDE DEHYDROGENASE FROM THE HALOPHILIC ARCHAEON, HALOFERAX-VOLCANII

A homology-modelled structure of dihydrolipoamide dehydrogenase from t he halophilic archaeon, Haloferax volcanii, has been generated using t he crystal structure of the enzyme from Pseudomonas fluorescens. Analy sis of the halophilic enzyme structure identified a potential K+-bindi ng 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 de hydrogenases. E423 is only present in the halophilic enzyme. Four site -directed mutations of the Haloferax dihydrolipoamide dehydrogenase ha ve been made (E423D, E423Q, E423S, and E423A) and the recombinant muta nts expressed and characterised. From an analysis of their kinetic pro perties, salt-dependent activities and thermal stabilities, it is conc luded that this sits has an important influence on the halophilicity o f the enzyme. The findings support the view that the arrangement and i nteraction of the negatively charged amino acids are as important as t he total net charge in determining the adaptation of proteins to high salt concentrations.