Mónica Camacho

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.

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.

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.

Purification and some properties of NAD+-dependent glutamate dehydrogenase from Halobacterium halobium

Abstract 1. 1. A NAD + -dependent glutamate dehydrogenase (EC 1.4.1.2.) was purified 126-fold from Halobacterium halobium . 2. 2. Activity and stability of the enzyme were affected by salt concentration. Maximum activity of the NADH-dependent reductive amination of 2-oxoglutarate occurs at 3.2 M NaCl and 0.8 M KCl, and the NAD + -dependent oxidative deamination of l -glutamate occurs at 0.9 M NaCl and 0.4 M KCl. The maximum activity is higher with Na + than with K + in the amination reaction while the reverse is true in the deamination reaction. 3. 3. The apparent K m values of the various substrates and coenzymes under optimal conditions were: 2-oxoglutarate, 20.2 mM; ammonium, 0.45 M; NADH, 0.07 mM; l -glutamate, 4.0 mM; NAD + , 0.30 mM. 4. 4. No effect of ADP or GTP on the enzyme activity was found. The purified enzyme was activated by some l -amino acids.

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.

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.

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.

ANALYSIS OF THE KINETIC MECHANISM OF HALOPHILIC NADP-DEPENDENT GLUTAMATE DEHYDROGENASE

The amination of 2-oxoglutarate catalyzed by NADP-specific glutamate dehydrogenase (EC 1.4.1.4, L-glutamate:NADP+ oxidoreductase (deaminating)) from Halobacterium halobium has been analyzed by initial rate, graphical analysis, and product and competitive inhibition studies. Initial rate and graphical analysis reveal that a B term (representing 2-oxoglutarate) is not statistically necessary for an initial rate equation. However, the absence of a B term does not distinguish between ordered and random binding of NADPH and ammonia. The patterns of product inhibition by NADP+ and L-glutamate, and competitive inhibition by hydroxylamine and succinate permit deduction of the kinetic mechanism as ordered, with NADPH, 2-oxoglutarate and ammonia added in that order, and L-glutamate release preceding NADP+ release.

Nitrate and nitrite removal from salted water by Haloferax mediterranei

Haloferax mediterranei is a denitrifying halophilic archaeon, able to assimilate nitrate or nitrite in the presence of oxygen by the assimilatory nitrate pathway. It can also grow in the presence of high nitrate or nitrite concentrations under anoxic conditions, using both nitrogen species as electron acceptors. In this study, the ability of H. mediterranei to remove high nitrate and nitrite concentrations from culture media has been demonstrated. This suggests that this haloarchaeon could be applied in water bioremediation processes to repair damage caused by anthropogenic activities. This could be beneficial in regions such as Comunidad Valenciana or Murcia (Spain), where the water tables contain high nitrate and nitrite concentrations due to fertiliser addition, and high salt concentrations due to marine intrusions.

Kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase

The kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase (EC 1.4.1.3) has been investigated at pH 9.0, 3 M NaCl and 40 degrees C in both directions, by initial rate and inhibition studies. The results of the initial rate studies indicate that the mechanism is sequential with respect to substrate addition. The inhibition patterns obtained with halophilic NAD+-glutamate dehydrogenase are not consistent with a simple ordered mechanism without modification. They can, however, be reconciled with this type of mechanism by postulating an appropriate abortive complex.