Juan Ferrer

T-type calcium channels contribute to colonic hypersensitivity in a rat model of irritable bowel syndrome.

The symptoms of irritable bowel syndrome (IBS) include significant abdominal pain and bloating. Current treatments are empirical and often poorly efficacious, and there is a need for the development of new and efficient analgesics aimed at IBS patients. T-type calcium channels have previously been validated as a potential target to treat certain neuropathic pain pathologies. Here we report that T-type calcium channels encoded by the CaV3.2 isoform are expressed in colonic nociceptive primary afferent neurons and that they contribute to the exaggerated pain perception in a butyrate-mediated rodent model of IBS. Both the selective genetic inhibition of CaV3.2 channels and pharmacological blockade with calcium channel antagonists attenuates IBS-like painful symptoms. Mechanistically, butyrate acts to promote the increased insertion of CaV3.2 channels into primary sensory neuron membranes, likely via a posttranslational effect. The butyrate-mediated regulation can be recapitulated with recombinant CaV3.2 channels expressed in HEK cells and may provide a convenient in vitro screening system for the identification of T-type channel blockers relevant to visceral pain. These results implicate T-type calcium channels in the pathophysiology of chronic visceral pain and suggest CaV3.2 as a promising target for the development of efficient analgesics for the visceral discomfort and pain associated with IBS.

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.

Occurrence of two different glutamate dehydrogenase activities in the halophilic bacterium Salinibacter ruber.

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 concentrations for both activity and stability. It catalyzed amination of 2-oxoglutarate with optimal activity in 3 M KCl at pH 8. No activating effect was found when NaCl was replaced by KCl. Only GDHII displayed activity in the deamination reaction of glutamate with an optimal pH of 9.5. Both enzymes were activated by certain amino acids (L-leucine, L-histidine, L-phenylalanine) and by nucleotides such as ADP or ATP. A low-molecular-mass cytoplasmic fraction was found to be a highly effective activator of GDHII in the presence of high NaCl concentrations.

NAD-glutamate dehydrogenase from Halobacterium halobium: inhibition and activation by TCA intermediates and amino acids.

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 enzyme activity. GTP, ATP, ADP and AMP did not affect the enzyme, so these effectors of bovine glutamate dehydrogenase do not play a role in the regulation of the halophilic enzyme. However, the halophilic enzyme was subject to strong inhibition by TCA intermediates. When measuring the initial rate of the reaction, the oxidative deamination of L-glutamate was inhibited by TCA metabolites such as: fumarate, oxalacetate, succinate and malate; by substrate analogues such as: NADP+, D-glutamate and glutarate; and by dicarboxylic compounds such as adipate. On the other hand, all the amino acids tested were activators of this enzyme, except the D-isomer of the substrate L-glutamate that acted as an inhibitor. The relative effectiveness of each inhibitor or activator (Ki or Ka values) was correlated with the dipole moment (mu), HOMO and LUMO molecular orbital energies, optimal distance between two carboxyl groups, and hydrophobicity. Compounds with high dipole moment acted as good activators while compounds with low dipole moment were inhibitors. We have also found that the best activators were amino acids with no polar lateral chain.

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.

NAD(P)+-glucose dehydrogenase from Haloferax mediterranei: kinetic mechanism and metal content

Abstract 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 that the mechanism is sequential with respect to substrate addition. The product inhibition patterns agree with an ordered binding of NADP+ and d -glucose, followed by an ordered release of gluconolactone and NADPH. The activity of Hf. mediterranei glucose dehydrogenase was markedly dependent on the concentration of metal ions. Inactivation by metal chelators and reactivation by certain divalent ions indicated that glucose dehydrogenase from Hf. mediterranei contains tightly bound metal ions which are essential for activity. Metal analyses demonstrated that the enzyme binds 3.6±0.3 mol of Zn(II)/mol of protein, which corresponds to the binding of two atoms of Zn(II) per subunit. Alignment of the N-terminal sequence of glucose dehydrogenase from Hf. mediterranei with medium chain zinc-containing dehydrogenases reveals a clear similarity between them, suggesting that glucose dehydrogenase from Hf. mediterranei belongs to this family.

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.

Denaturation studies by fluorescence and quenching of thermophilic protein NAD+-glutamate dehydrogenase from Thermus thermophilus HB8.

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 thermophilic glutamate dehydrogenase (t-GDH) have been analyzed. Native t-GDH presents the maximum emission at 338 nm. The denaturation process is accompanied by an exposure to the solvent of the tryptophan residues, as manifested by the red shift of the emission maximum. Fluorescence quenching by external quenchers, KI and acrylamide, has also been carried out.

Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum.

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 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.