The structure of glucose dehydrogenase from the extreme halophile Haloferax mediterranei has been solved at 1.6-Å resolution under crystallization conditions which closely mimic the ''in vivo'' intracellular environment. The decoration of the enzyme's surface with acidic residues is only partially neutralized by bound potassium counterions, which also appear to play a role in substrate binding. The surface shows the expected reduction in hydrophobic character, surprisingly not from changes associated with the loss of exposed hydrophobic residues but rather arising from a loss of lysines consistent with the genome wide-reduction of this residue in extreme halophiles. The structure reveals a highly ordered, multilayered solvation shell that can be seen to be organized into one dominant network covering much of the exposed surface accessible area to an extent not seen in almost any other protein structure solved. This finding is consistent with the requirement of the enzyme to form a protective shell in a dehydrating environment.Archaea ͉ x-ray structure ͉ water structure ͉ hydrophobic surface ͉ surface lysines

The nitrogen cycle (N-cycle), principally supported by prokaryotes, involves different redox reactions mainly focused on assimilatory purposes or respiratory processes for energy conservation. As the N-cycle has important environmental implications, this biogeochemical cycle has become a major research topic during the last few years. However, although N-cycle metabolic pathways have been studied extensively in Bacteria or Eukarya, relatively little is known in the Archaea. Halophilic Archaea are the predominant microorganisms in hot and hypersaline environments such as salted lakes, hot springs or salted ponds. Consequently, the denitrifying haloarchaea that sustain the nitrogen cycle under these conditions have emerged as an important target for research aimed at understanding microbial life in these extreme environments.The haloarchaeon Haloferax mediterranei was isolated 20 years ago from Santa Pola salted ponds (Alicante, Spain). It was described as a denitrifier and it is also able to grow using NO 3 -, NO 2 -or NH 4 + as inorganic nitrogen sources. This review summarizes the advances that have been made in understanding the N-cycle in halophilic archaea using Hfx mediterranei as a haloarchaeal model. The results obtained show that this microorganism could be very attractive for bioremediation applications in those areas where high salt, nitrate and nitrite concentrations are found in ground waters and soils.

An NAD(P)-glucose dehydrogenase from the extremely halophific Archaeon, Haloferax mediterranei, has been purified to electrophoretic homogeneity. The purified enzyme has been characterised with respect to its cofactor specificity, subunit composition and its salt and thermal stability. The N-terminal amino acid sequence has been determined and N-terminus alignment with sequences of other glucose dehydrogenases shows that the halophilic enzyme most closely resembles the NAD(P)-linked glucose dehydrogenase from the thermophilic Archaeon Thermoplasma acidophilum. However, the halophilic glucose dehydrogenase appears to be a dimeric protein, in contrast to the tetrameric enzyme from the thermophile.

The halophilic archaeon Haloferax mediterranei is able to grow in a minimal medium containing ammonium acetate as a carbon and nitrogen source. When this medium is enriched with starch, alpha-amylase activity is excreted to the medium in low concentration. Here we report methods to concentrate and purify the enzyme. The relative molecular mass of the enzyme, determined by gel filtration, is 50 +/- 4 kDa, and on SDS-PAGE analysis a single band appeared at 58 kDa. These results indicated that the halophilic alpha-amylase is a monomeric enzyme. The enzyme showed a salt requirement for both stability and activity, being stable from 2 to 4 M NaCl, with maximal activity at 3 M NaCl. The enzyme displayed maximal activity at pHs from 7 to 8, and its optimal temperature was in a range from 50 degrees C to 60 degrees C. The results also implicated several prototropic groups in the catalytic reaction.

Despite being the subject of intensive investigations, many aspects of the mechanism of the zinc-dependent medium chain alcohol dehydrogenase (MDR) superfamily remain contentious. We have determined the high-resolution structures of a series of binary and ternary complexes of glucose dehydrogenase, an MDR enzyme from Haloferax mediterranei. In stark contrast to the textbook MDR mechanism in which the zinc ion is proposed to remain stationary and attached to a common set of protein ligands, analysis of these structures reveals that in each complex, there are dramatic differences in the nature of the zinc ligation. These changes arise as a direct consequence of linked movements of the zinc ion, a zinc-bound bound water molecule, and the substrate during progression through the reaction. These results provide evidence for the molecular basis of proton traffic during catalysis, a structural explanation for pentacoordinate zinc ion intermediates, a unifying view for the observed patterns of metal ligation in the MDR family, and highlight the importance of dynamic fluctuations at the metal center in changing the electrostatic potential in the active site, thereby influencing the proton traffic and hydride transfer events.MDR family ͉ structure ͉ zinc metalloenzyme ͉ reaction mechanism Z inc-dependent enzymes catalyze many important cellular processes (1); yet, despite this, the role played by the zinc ion in catalysis is not completely understood, partly because zinc is silent in a range of useful spectroscopic techniques, such as EPR and optical spectroscopy. One of the best characterized zinccontaining enzyme families is the Zn-dependent medium chain alcohol dehydrogenase superfamily (MDR), which catalyzes the oxidation of primary or secondary alcohols to the corresponding aldehydes or ketones using NAD(P) ϩ as a cofactor (2).The structures of many MDR family members have been determined with that of liver alcohol dehydrogenase (LADH) as the prototypical member (3). These studies have shown that the subunit of these enzymes is constructed from two domains, a nucleotide binding domain and a catalytic domain (4), with the essential zinc ion located deep in the cleft between them. The ligands to the zinc are provided by residues from the catalytic domain and, in the absence of substrate, the zinc is coordinated to the side chains of three well conserved residues, which in LADH are Cys-46, His-67, and Cys-174 (2). The tetrahedral coordination shell of the zinc is completed by a water molecule (2).In the classical text book mechanism for this enzyme family (5), this zinc-bound water molecule is suggested to be displaced by the incoming hydroxyl group of the alcohol on substrate binding, to leave the zinc ion coordinated to the substrate and the same three protein ligands as in the apo enzyme. The redox reaction requires the net removal of two hydrogen atoms from the substrate and is thought to proceed via proton loss from the substrate hydroxyl to bulk solvent by using a proton relay system to form an alkoxide intermedia...