Ka. Jolley et al., SITE-DIRECTED MUTAGENESIS AND HALOPHILICITY OF DIHYDROLIPOAMIDE DEHYDROGENASE FROM THE HALOPHILIC ARCHAEON, HALOFERAX-VOLCANII, European journal of biochemistry, 248(2), 1997, pp. 362-368
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.