Julia Esclapez

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.

Analysis of acidic surface of Haloferax mediterranei glucose dehydrogenase by site‐directed mutagenesis

Generally, halophilic enzymes present a characteristic amino acid composition, showing an increase in the content of acidic residues and a decrease in the content of basic residues, particularly lysines. The latter decrease appears to be responsible for a reduction in the proportion of solvent‐exposed hydrophobic surface. This role was investigated by site‐directed mutagenesis of glucose dehydrogenase from Haloferax mediterranei, in which surface aspartic residues were changed to lysine residues. From the biochemical analysis of the mutant proteins, it is concluded that the replacement of the aspartic residues by lysines results in slightly less halotolerant proteins, although they retain the same enzymatic activities and kinetic parameters compared to the wild type enzyme.

Stability and Enzymatic Studies of Glucose Dehydrogenase from the Archaeon Haloferax mediterranei in reverse micelles

Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant glucose dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w0=[H2O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-glucose to D-glucono-1,5-lactone using NADP+ as coenzyme for the glucose dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.

Crystallization and preliminary X-ray analysis of glucose dehydrogenase from Haloferax mediterranei.

Glucose dehydrogenase (E.C. 1.1.1.47; GlcDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized by the addition of 8 M urea and refolded by rapid dilution. The protein has been purified by conventional techniques and crystallized by the hanging-drop vapour-diffusion method using sodium citrate as the precipitant. Two crystal forms representing the free enzyme and the binary complex with NADP(+) grow under these conditions. Crystals of form I diffract to beyond 3.5 A resolution and belong to the hexagonal space group P622, with unit-cell parameters a = b = 89.1, c = 214.6 A, alpha = beta = 90, gamma = 120 degrees. Crystals of form II diffract to greater than 2.0 A and belong to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 61.8, b = 110.9, c = 151.7 A, alpha = beta = gamma = 90 degrees. Calculated values for V(M) and consideration of the packing for both crystal forms suggests that the asymmetric units in both crystal forms contain a monomer.

Crystallization and preliminary X-ray analysis of binary and ternary complexes of Haloferax mediterranei glucose dehydrogenase

Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse the significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.

Effects of nitrogen sources on the nitrate assimilation in Haloferax mediterranei: growth kinetics and transcriptomic analysis

The haloarchaeon Haloferax mediterranei is able to grow in a defined culture media not only in the presence of inorganic nitrogen salt but also with amino acid as the sole nitrogen source. Assimilatory nitrate and nitrite reductases, respectively, catalyze the first and second reactions. The genes involved in this process are nasA, which encodes nitrate reductase and is found within the operon nasABC, and nasD, which encodes nitrite reductase. These genes are subjected to transcriptional regulation, being repressed in the presence of ammonium and induced with either nitrate or nitrite. This type of regulation has also been described when the amino acids are used as nitrogen source in the minimal media. Furthermore, it has been observed that the microorganism growth depends on nitrogen source, obtaining the lowest growth rate in the presence of nitrate and aspartate. In this paper, we present the results of a comparative study of microorganism growth and transcriptomic analysis of the operon nasABC and gene nasD in different nitrogen sources. The results are the first ever produced in relation to amino acids as nitrogen sources within the Halobacteriaceae family.

Anaerobic Metabolism in Haloferax Genus

A number of species of Haloferax genus (halophilic archaea) are able to grow microaerobically or even anaerobically using different alternative electron acceptors such as fumarate, nitrate, chlorate, dimethyl sulphoxide, sulphide and/or trimethylamine. This metabolic capability is also shown by other species of the Halobacteriaceae and Haloferacaceae families (Archaea domain) and it has been mainly tested by physiological studies where cell growth is observed under anaerobic conditions in the presence of the mentioned compounds. This work summarises the main reported features on anaerobic metabolism in the Haloferax, one of the better described haloarchaeal genus with significant potential uses in biotechnology and bioremediation. Special attention has been paid to denitrification, also called nitrate respiration. This pathway has been studied so far from Haloferax mediterranei and Haloferax denitrificans mainly from biochemical point of view (purification and characterisation of the enzymes catalysing the two first reactions). However, gene expression and gene regulation is far from known at the time of writing this chapter.

Anaerobic Metabolism in Haloferax Genus: Denitrification as Case of Study

A number of species of Haloferax genus (halophilic archaea) are able to grow microaerobically or even anaerobically using different alternative electron acceptors such as fumarate, nitrate, chlorate, dimethyl sulphoxide, sulphide and/or trimethylamine. This metabolic capability is also shown by other species of the Halobacteriaceae and Haloferacaceae families (Archaea domain) and it has been mainly tested by physiological studies where cell growth is observed under anaerobic conditions in the presence of the mentioned compounds. This work summarises the main reported features on anaerobic metabolism in the Haloferax, one of the better described haloarchaeal genus with significant potential uses in biotechnology and bioremediation. Special attention has been paid to denitrification, also called nitrate respiration. This pathway has been studied so far from Haloferax mediterranei and Haloferax denitrificans mainly from biochemical point of view (purification and characterisation of the enzymes catalysing the two first reactions). However, gene expression and gene regulation is far from known at the time of writing this chapter.

Transcriptional profiles of Haloferax mediterranei based on nitrogen availability

The haloarchaeon Haloferax mediterranei is able to grow in the presence of different inorganic and organic nitrogen sources by means of the assimilatory pathway under aerobic conditions. In order to identify genes of potential importance in nitrogen metabolism and its regulation in the halophilic microorganism, we have analysed its global gene expression in three culture media with different nitrogen sources: (a) cells were grown stationary and exponentially in ammonium, (b) cells were grown exponentially in nitrate, and (c) cells were shifted to nitrogen starvation conditions. The main differences in the transcriptional profiles have been identified between the cultures with ammonium as nitrogen source and the cultures with nitrate or nitrogen starvation, supporting previous results which indicate the absence of ammonium as the factor responsible for the expression of genes involved in nitrate assimilation pathway. The results have also permitted the identification of transcriptional regulators and changes in metabolic pathways related to the catabolism and anabolism of amino acids or nucleotides. The microarray data was validated by real-time quantitative PCR on 4 selected genes involved in nitrogen metabolism. This work represents the first transcriptional profiles study related to nitrogen assimilation metabolism in extreme halophilic microorganisms using microarray technology.

Cu-NirK from Haloferax mediterranei as an example of metalloprotein maturation and exportation via Tat system

The green Cu-NirK from Haloferax mediterranei (Cu-NirK) has been expressed, refolded and retrieved as a trimeric enzyme using an expression method developed for halophilic Archaea. This method utilizes Haloferax volcanii as a halophilic host and an expression vector with a constitutive and strong promoter. The enzymatic activity of recombinant Cu-NirK was detected in both cellular fractions (cytoplasmic fraction and membranes) and in the culture media. The characterization of the enzyme isolated from the cytoplasmic fraction as well as the culture media revealed important differences in the primary structure of both forms indicating that Hfx. mediterranei could carry out a maturation and exportation process within the cell before the protein is exported to the S-layer. Several conserved signals found in Cu-NirK from Hfx. mediterranei sequence indicate that these processes are closely related to the Tat system. Furthermore, the N-terminal sequence of the two Cu-NirK subunits constituting different isoforms revealed that translation of this protein could begin at two different points, identifying two possible start codons. The hypothesis proposed in this work for halophilic Cu-NirK processing and exportation via the Tat system represents the first approximation of this mechanism in the Halobacteriaceae family and in Prokarya in general.