ADAPTATION TO EXTREME SALT ENVIRONMENT: THE STRUCTURE OF DIHYDROFOLATE REDUCTASE FROM HALOFERAX VOLCANII. Ursula Pieper, Geeta Kapadia, Moshe Mevarech and Osnat Herzberg, Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville MD 20850, USA

Haloferax volcanii is a halophilic archaeon, native in the dead sea, that adapted to high salt concentrations by accumulating up to 4M KCl inside the cytoplasmic membrane. Amino acid sequence analysis of a number of halophilic proteins reveals an enrichment of negatively charged amino acids which have been proposed to solvate the protein more efficiently.

We present here the crystal structure of dihydrofolate reductase from Haloferax volcanii (h-DHFR), the first structure of a protein originated from this organism and the third structure of a halophilic protein. The structure has been determined by molecular replacement using as a search model the superpositions of seven DHFR coordinate sets available in the PDB. Subsequently, the structure has been refined at 2.6 Å resolution to an R-value of 0.18.

The overall structure of h-DHFR is very similar to the other known DHFR structures. The largest differences occur in one helix and a loop region involved in NADP binding. There are no obvious features that correlate with the enzyme functioning at high salt. This is in contrast to the report of unusual charge clusters found in malate dehydrogenase from Haloarcula marismortui. For providing structural basis to the functional behavior, we argue that it is not the ability to function in high salt that needs explaining, because many other mesophilic proteins function well at high salt. Rather, the impaired function at low salt should be rationalized. In that respect, h-DHFR may not be a genuine extreme halophilic enzyme, because it is active at rather low salt concentrations. Nevertheless a number of subtle differences in both the amino acid composition and tertiary structure are observed and together they may account for the halophilic behavior.