María José Bonete

Extreme halophilic enzymes in organic solvents

The use of halophilic extremozymes in organic media has been limited by the lack of enzymological studies in these media. To explore the behaviour of these extremozymes in organic media, different approaches have been adopted, including the dispersal of the lyophilised enzyme or the use of reverse micelles. The use of reverse micelles in maintaining high activities of halophilic extremozymes under unfavourable conditions could open new fields of application such as the use of these enzymes as biocatalysts in organic media.

Glucose dehydrogenase from the halophilic Archaeon Haloferax mediterranei: Enzyme purification, characterisation and N-terminal sequence

An NAD(P)‐glucose dehydrogenase from the extremely halophilic 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.

Nitrogen metabolism in haloarchaea

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 NO3-, NO2- or NH4+ 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.

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

Abstract 1. 1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme. 2. 2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium. 3. 3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate. 4. 4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M. 5. 5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme. 6. 6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.

Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily

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.

Operation of glyoxylate cycle in halophilic archaea: presence of malate synthase and isocitrate lyase in Haloferax volcanii

The occurrence of the glyoxylate cycle has not previously been demonstrated in any of the Archaea. In halophilic archaea, only isocitrate lyase activity has been detected. The halophilic archaeon Haloferax volcanii was tested for the presence of the other key enzyme of this pathway, malate synthase. High activities of this enzyme were detected when the carbon source was acetate. Both glyoxylate cycle key enzymes, isocitrate lyase and malate synthase, from Hf. volcanii were purified and characterized.

Sequencing, phylogenetic and transcriptional analysis of the glyoxylate bypass operon (ace) in the halophilic archaeon Haloferax volcanii

The glyoxylate cycle occurs in the three domains of living organisms: Eukarya, Bacteria and Archaea. We have isolated and sequenced the ace (acetate assimilation) gene operon, comprising the glyoxylate cycle key enzymes isocitrate lyase and malate synthase genes (icl or aceA and ms or aceB), from the halophilic archaeon Haloferax volcanii. This is the first time that these genes are sequenced in an organism from the domain Archaea. Phylogenetic analysis of the sequenced genes revealed that isocitrate lyase shows a significant identity with isocitrate lyases from Eukarya and Bacteria, but it is not more closely related to eukaryal or bacterial enzymes, and that malate synthase from H. volcanii has very little identity with any other known protein. This enzyme forms a new class of malate synthases. Transcriptional analysis indicated that both genes are cotranscribed in a single 2.7 kb mRNA molecule. The genes were transcribed only when acetate was the carbon source, indicating transcriptional regulation. Two sets of palindromic sequences were found in the promoter region, possibly involved in binding of transcriptional regulators (repressors and/or activators).

Respiratory nitrate and nitrite pathway in the denitrifier haloarchaeon Haloferax mediterranei

Haloferax mediterranei cells are able to use high nitrate or nitrite concentrations as electron acceptors under anoxic conditions. The nar operon, which has eight open reading frames, has been sequenced and its regulation has been characterized at the transcriptional level. The narG and narH genes encode the Nar (respiratory nitrate reductase) catalytic subunit (NarG) and the electron transfer Nar subunit (NarH) respectively. Nar has been purified and characterized in vitro. This characterization has included protein-film voltammetry and preliminary EPR studies.

Carotenoids from Haloarchaea and Their Potential in Biotechnology.

The production of pigments by halophilic archaea has been analysed during the last half a century. The main reasons that sustains this research are: (i) many haloarchaeal species possess high carotenoids production availability; (ii) downstream processes related to carotenoid isolation from haloarchaea is relatively quick, easy and cheap; (iii) carotenoids production by haloarchaea can be improved by genetic modification or even by modifying several cultivation aspects such as nutrition, growth pH, temperature, etc.; (iv) carotenoids are needed to support plant and animal life and human well-being; and (v) carotenoids are compounds highly demanded by pharmaceutical, cosmetic and food markets. Several studies about carotenoid production by haloarchaea have been reported so far, most of them focused on pigments isolation or carotenoids production under different culture conditions. However, the understanding of carotenoid metabolism, regulation, and roles of carotenoid derivatives in this group of extreme microorganisms remains mostly unrevealed. The uses of those haloarchaeal pigments have also been poorly explored. This work summarises what has been described so far about carotenoids production by haloarchaea and their potential uses in biotechnology and biomedicine. In particular, new scientific evidence of improved carotenoid production by one of the better known haloarchaeon (Haloferax mediterranei) is also discussed.

Salt-dependent studies of NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii

The salt-dependent stability of recombinant dimeric isocitrate dehydrogenase [ICDH; isocitrate: NADP oxidoreductase (decarboxylating), EC 1.1.1.42] from the halophilic archaeon Haloferax volcanii (Hv) was investigated in various conditions. Hv ICDH dissociation/deactivation was measured to probe the respective effect of anions and cations on stability. Surprisingly, enzyme stability was found to be mainly sensitive to cations and very little (or not) sensitive to anions. Divalent cations induced a strong shift of the active/inactive transition towards low salt concentration. A high resistance of Hv ICDH to chemical denaturation was also found. The data were analysed and are discussed in the framework of the solvation stability model for halophilic proteins.