Juan Ferrer

University of Alicante | UA · Department of Agrochemistry and Biochemistry

The selectivity of the sodium channel has been the subject of numerous experimental and theoretical studies. In this work, this problem is approached from a theoretical point of view based on a model built from the Selective Filter (SF) of the open structure of the voltage-activated channel of the bacterium Magnetococcus marinus. This approach has...

Numerous theoretical and experimental studies have attempted to determine the mechanisms of bacterial potassium channel selectivity (KcsA). However, there are still different aspects that remain uncovered. In this paper, we have built a model based on a selective filter (SF) for the KcsA, taking into account its structure and calculating the system...

Saproxylic insect communities inhabiting tree hollows in Mediterranean forests depend on a combination of physical characteristics and interactions occurring between community member species. Despite the need to preserve these organisms, little is known about their interrelationships, in particular those relationships between saproxylic insects and...

The competition between \(\hbox {Na}^+\) and \(\hbox {K}^+\) with a protein for water was investigated by using Density Functional Theory (DFT) calculations. The optimized potential energy curves have been made in the DFT, together with balanced basis sets of split valence Def2-SV(P). Initially, calculations were done in order to know the organizat...

The halophilic enzyme D-2-hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei is found to be an oligomeric enzyme composed of two identical subunits. Fluorescence spectra of native and denatured protein and effect of denaturants such as urea and guanidine hydrochloride on enzyme activity of halophilic D-2-hydroxyacid dehydrogenase have b...

Structural analysis of glucose dehydrogenase from Haloferax mediterranei revealed that the adenosine 2′-phosphate of NADP+ was stabilized by the side chains of Arg207 and Arg208. To investigate the structural determinants for coenzyme specificity, several mutants involving residues Gly206, Arg207 and Arg208 were engineered and kinetically character...

The kinetic mechanism of NADP-glutamate dehydrogenase (EC 1.4.1.4) from the Archaeon Haloferax mediterranei was studied in 3 M KC1 and in glycerol. Haloferax mediterranei is a halophilic organism requiring 20-25% NaCl for optimal growth, so its enzymes are stabilised by high salt concentrations. We have replaced the salt by 20% (v/v) glycerol in or...

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 re...

D-2-hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized in 8 M urea and refolded by rapid dilution. The protein was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate or PEG 3350 as precipitant. Two crystal forms representing the free enzyme...

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 star...

El presente trabajo ha sido subencionado en parte por los proyectos: BI093-0660-CO4-03 CICYT y GV-1170793 Generalitat Valenciana.

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...

A gene encoding a new D-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple...

The NAD-dependent glutamate dehydrogenase (GDH) gene from the halophilic archaeon Haloferax mediterranei has been cloned. The analysis of the nucleotide sequence revealed an open reading frame of 1323 bp that encodes a NAD-GDH. The amino acid sequence displayed high homology with those from other sources, especially the highly conserved residues in...

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 als...

An NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8 was purified 350-fold by a several-step procedure involving Blue-Sepharose chromatography. The native protein had a molecular mass of approximately 289 kDa, and consisted of six subunits with a molecular mass of 48 kDa each. The optimum pH for the deaminating reaction was 8.0. Th...

who generously supported the meeting. Meeting

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. mediterr...

Nitrate is an important inorganic nitrogen source for plants and microorganisms. The physiology, enzymology and genetics of nitrate assimilation have been well studied in plants and bacteria (Campbell 1996; Lin and Stewart 1998); nonetheless, little is known at the biochemical, genetic or structural level of this process in halophilic Archaea. Nitr...

Salinibacter ruber, an extremely halophilic member of the domain Bacteria, has two different cytoplasmic glutamate dehydrogenase activities, marked as GDHI and GDHII. GDHI showed a strong dependence on high salt concentrations for stability, but not for activity, displaying maximal activity in the absence of salts. GDHII depended on high salt conce...

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 t...

Fluorescence techniques have been used to study the structural characteristics of many proteins. The thermophilic enzyme NAD-glutamate dehydrogenase from Thermus thermophilus HB8 is found to be a hexameric enzyme. Fluorescence spectra of native and denatured protein and effect of denaturants as urea and guanidine hydrochloride on enzyme activity of...

The amplified fragment length polymorphism (AFLP) technique was applied to identify palm varieties. Fluorescence labelled primers were used in selective amplifications and the amplified fragments were detected on capillary gel electrophoresis using an automated DNA sequencer with the analysis fragment option. This is a rapid and efficient technique...

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...

The first gene encoding a glucose dehydrogenase (GDH) from a halophilic organism has been sequenced. Amino acid sequence alignments of GDH from Haloferax mediterranei show a high degree of homology with the thermoacidophilic GDHs and with other enzymes from the medium chain dehydrogenase/reductase family. Heterologous overexpression using the mesop...

An NAD-dependent D-2-hydroxyacid dehydrogenase (EC 1.1.1.) was isolated and characterized from the halophilic Archaeon Haloferax mediterranei. The enzyme is a dimer with a molecular mass of 101.4 +/- 3.3 kDa. It is strictly NAD-dependent and exhibits its highest activity in 4 M NaCl. The enzyme is characterized by a broad substrate specificity 2-ke...

The kinetic mechanism and metal content of Haloferax mediterranei NAD(P)+-glucose dehydrogenase have been investigated. The kinetic mechanism has been determined by initial rate and inhibition studies. Initial velocity studies were performed with d-glucose as well as with the alternative substrate d-xylose, with NADP+ as coenzyme. The results show...

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at...

Fluorescence techniques have been used to study the structural characteristics of many proteins. The halophilic enzyme NADP-glutamate dehydrogenase from Haloferax mediterranei is found to be a hexameric enzyme composed of identical subunits. Fluorescence spectra of native and denatured halophilic and bovine glutamate dehydrogenase (h-GDH and b-GDH)...

An NADP(H)-specific glutamate dehydrogenase of Haloferax mediterranei has been purified to apparent homogeneity and characterised. The purified enzyme was stabilized by glycerol in absence of salt. Glutamate dehydrogenase from Hf. mediterranei is a hexameric enzyme with a native molecular mass of 320 kDa composed of monomers each with a molecular m...

An NADP(H)-specific glutamate dehydrogenase of Haloferax mediterranei has been purified to apparent homogeneity and characterised. The purified enzyme was stabilized by glycerol in absence of salt. Glutamate dehydrogenase from Hf. mediterranei is a hexameric enzyme with a native molecular mass of 320 kDa composed of monomers each with a molecular m...

A variety of metabolites have been found to elicit a form of inhibition or activation on an NAD-specific glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from Halobacterium halobium. The purified halophilic enzyme was tested with several compounds known to be allosteric modifiers of mammalian glutamate dehydrogenases to determine their effects on enzy...