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The glutathione biosynthesis in the psychrophile Pseudoalteromonas haloplanktis
No full textReduced glutathione (GSH), together with its oxidized form (GSSG), is the most effective antioxidant system responsible for controlling the cellular redox state. Its biosynthesis from glutamate, cysteine and glycine, normally requires two enzymes. Indeed, γ-glutamyl-cysteine ligase (GshA) forms γ-glutamyl-cysteine, whereas glutathione synthetase (GshB) leads to the formation of GSH. In the genome of Pseudoalteromonas haloplanktis, a psychrophilic eubacterium isolated from Antarctic sea water, two genes coding for GshA (PhGshA-I and PhGshA-II ) and one gene for GshB (PhGshB) were putatively identified. The study of the biochemical properties of these enzymes was addressed with an appropriate heterologous expression system, thus leading to the production of the recombinant forms of PhGshB and PhGshA-II (rPhGshB and rPhGshA-II), purified by affinity chromatography.
The first enzyme investigated was rPhGshB. Its purification was achieved either in the absence or in the presence of β-mercaptoethanol. The study of its molecular properties showed that, when purified in the presence of β-mercaptoethanol, rPhGshB underwent a covalent modification; however, this modification did not significantly affect its biochemical properties. The molecular mass of rPhGshB in denaturing conditions was 36 kDa, corresponding to the mass of the PhGshB monomer; in non denaturing conditions the mass determined by gel-filtration ranged between 74 and 136 kDa, respectively, values corresponding to a dimeric or tetrameric organization. The different behavior depended on the enzyme concentration and the data suggested that at higher concentrations the enzyme formed an unstable tetramer that at lower concentrations was converted into a dimeric and more stable form.
To study the activity of rPhGshB, a new method for direct determination was developed, based on the hydrolysis of the radioactive substrate [γ32P] ATP; in fact, the synthesis of GSH catalyzed by GshB is coupled to the hydrolysis of ATP. The ATPase activity of rPhGshB required the presence of the other two substrates, glycine and γ-glutamylcysteine (γ-Glu-Cys). rPhGshB showed its maximum activity in the 7.4-8.2 pH range. The enzyme activity required also the presence of a divalent cation and at 5 mM Mg++ reached its maximum.
The kinetic parameters of rPhGshB at 15°C, the optimum value for growth of P. haloplanktis, were determined. The enzyme showed a comparable affinity for ATP and γ-Glu-Cys (Km = 0.26 mM and 0.25 mM, respectively), whereas a lower affinity was determined for glycine (Km = 0.75 mM). The comparison of these data with those of the corresponding enzyme from other sources showed the remarkable similarity with Escherichia coli; a similar lower affinity for glycine compared to the other two substrates was found in the other sources. Enzyme inhibition studies showed that GSSG is an inhibitor of rPhGshB. The effect of temperature on the kinetic parameters of rPhGshB was analysed in the temperature range 10-30°C. The data showed that rPhGshB is already active at 10°C and its Vmax significantly increased with temperature up to 25°C; on the other hand, in the temperature interval considered, a minimum variation of Km was observed. The kcat values were then used to draw an Arrhenius plot and in the range of linearity (10-25°C) an activation energy of 75 kJ/mol was determined, a value quite high for a psychrophilic enzyme.
The thermostability of rPhGshB was analyzed using a thermal inactivation profile and an extrapolated half-life of 10 min at 50.5°C was derived. This value was slightly high for a psychrophilic enzyme, although not unusual for enzymes involved in the control of the cellular redox state. A value of 208 kJ/mol was calculated for the energy of activation of the heat inactivation process; this value was intermediate between those usually obtained for psychrophilic and mesophilic enzymes, respectively.
Finally, the development of a new crystallization technique allowed the obtainment of crystal forms of rPhGshB, useful for the determination of the three-dimensional structure of the enzyme by X-ray diffraction.
The biochemical characterization of rPhGshA-II was started, using the same assay adopted for rPhGshB. Preliminary data obtained showed that the enzyme activity of rPhGshA-II requires a reducing agent in the reaction mixture; furthermore, the presence of the other two substrates glutamate and cysteine, is also required. The activity of rPhGshA-II needed a higher concentration of Mg++ (10-20 mM) compared to that already determined for rPhGshB. When the characterization of the molecular and biochemical properties of PhGshA-II will be completed, it will be possible to reconstitute in vitro for the first time the system for the biosynthesis of glutathione in a psychrophilic organism
Phenylmethanesulfonyl fluoride inactivates an archaeal superoxide dismutase by chemical modification of a specific tyrosine residue Cloning, sequencing and expression of the gene coding for Sulfolobus solfataricus superoxide dismutase
No full textThe gene encoding the superoxide dismutase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsSOD) was cloned and sequenced and its expression in Escherichia coli obtained. The chemicophysical properties of the recombinant SsSOD were identical with those of the native enzyme. The recombinant SsSOD possessed a covalent modification of Tyr41, already observed in native SsSOD [Ursby, T., Adinolfi, B.S., Al-Karadaghi, S., De Vendittis, E. & Bocchini, V. (1999) J. Mol. Biol. 286, 189± 205]. HPLC analysis of SsSOD samples prepared from cells treated or not with phenylmethanesulfonyl fluoride (PhCH2SO2F), a protease inhibitor routinely added during the preparation of cell-free extracts, showed that the modification was caused by PhCH2SO2F. Refinement of the crystal model of SsSOD confirmed that a phenylmetha- nesulfonyl moiety was attached to the hydroxy group of Tyr41. PhCH2SO2F behaved as an irreversible inactivator of SsSOD; in fact, the specific activity of both native and recombinant enzyme decreased as the percentage of modification increased. The covalent modification caused by PhCH2SO2F reinforced the heat stability of SsSOD. These results show that Tyr41 plays an important role in the enzyme activity and the maintenance of the structural architecture of SsSOD
Biochemical and functional characterization of superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis
No full textCrystallization and preliminary X-ray diffraction studies of a psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis
No full textThe Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high activity even at low temperature. Using hanging-drop vapour-diffusion technique, PhSOD has been successfully crystallized in two different crystal forms. Both crystal forms are monoclinic with space group P21 and diffract to 2.1 Å resolution. Form I has unit cell parameters a=45.49Ǻ b=103.63 Ǻ c=50.36 Ǻ =108.2° and contains a homodimer in the asymmetric unit. Form II has unit-cell parameters a=50.48Ǻ b=103.78Ǻ c=90.26Ǻ =103.8° and an asymmetric unit containing two PhSOD homodimers. Structure determination has been achieved using molecular replacement. The crystallographic study of this cold-adapted enzyme could contribute to the understanding of the molecular mechanisms of cold-adaptation and of the high catalytic efficiency at low temperatur
The elongation factor G carries a catalytic site for GTP hydrolysis, which is revealed by using 2-propanol in the absence of ribosomes
No full textIn the absence of ribosomal particles, elongation factor G (EF-G) promotes very little GTP hydrolysis. After the addition of some aliphatic alcohols to EF-G, the rate of nucleotide cleavage was significantly increased and GTPase activity was easily detectable. The highest stimulation, nearly 16-fold, occurred with 2-propanol at a 20% (v/v) concentration. The reaction showed the characteristics of an enzymatic catalysis, but the rate was three orders of magnitude lower than that of the ribosome-dependent EF-G GTPase activity. Striking similarities between the two activities indicated that the catalysis stimulated by the alcohol was due to EF-G itself. We found that EF-G GTPase activity in the presence of 2-propanol displayed an absolute specificity for GTP as in the presence of ribosomes; the two activities copurified to a constant ratio and exhibited coincident chromatographic and electrophoretic patterns; the temperature for the half-inactivation of EF-G was 59.3 degrees C for both GTPase systems, as well as the kinetic constant for the thermal inactivation process which was found to be 0.05 min-1; and the Km for the GTP in the presence of 2-propanol (59 microM) was similar to that found in the presence of ribosomes. These results indicate that the EF-G molecule carries a catalytic site for GTP hydrolysis, which in the absence of ribosomal particles is activated by an appropriate alcohol/water surrounding medium
Effect of solvent compositon on the critical micelle concentration of surfactants used in biological work
No full textCloning and sequencing of the gene encoding thermostable elongation factor 2 in Sulfolobus solfataricus
No full textThe gene (aEF-2) coding for the translation elongation factor 2 (aEF-2) in the thermoacidophilic archaebacterium, Sulfolobus solfataricus, has been cloned and sequenced. The deduced primary structure of aEF-2 is composed of 735 amino acids (aa), excluding the Met start residue. There are no Cys residues and the calculated M(r) is 81,699. In the coding region of aEF-2, the high A + T content greatly influences the codon usage. From the alignment of the primary structure of aEF-2 with that of the analogous factors from the three kingdoms, aa identities were derived. The greatest identity (82%) was found with EF-2 from Sulfolobus acidocaldarius; lower values were observed with other archaebacterial EF-2 (45-47%), eukaryotic EF-2 (38-40%) and with the functional eubacterial analogue EF-G (28-31%). aEF-2 possesses the consensus sequences required for a GTP-binding protein and the four regions which are supposed to be involved in the functional regulation of EF-2/EF-G. These data should have phylogenetic implications
Enzymes involved in the oxidative stress in Sulfolobus solfataricus
No full textThe present article summarises our recent work carried out on enzymes involved in the oxidative stress in the hyperthermophilic archaeon Sulfolobus solfataricus. The functional role of three key enzymes, superoxide dismutase (SOD), thioredoxin reductase (TrxR), and NADH oxidase (NOX) has been investigated. The study included a detailed structure–function relationship on these ubiquitous proteins through the characterisation of their molecular and functional properties, the comparison with the features of the corresponding enzymes isolated from taxonomically different sources, and a mutagenic analysis on some specific amino acid residues. S. solfataricus SOD belongs to the family of Fe- and Mn-SOD and possesses a very compact homotetrameric structure, responsible for its great heat resistance. The mutagenic analysis regarded two interacting residues of the active site, namely a conserved tyrosine and a semi–invariant histidine. The data on the tyrosine residue point to its relevance in the catalytic mechanism of superoxide dismutation, and indicate its high reactivity towards modifying agents; vice versa, the histidine residue is important for the structural architecture of the active site. S. solfataricus TrxR, an enzyme previously isolated for its ability to oxidise NADH, belongs to the family of class II pyridine nucleotide–disulphide oxidoreductases. It possesses two cysteine residues in the active site, forming a disulphide bridge essential for both reductase and NADH oxidase activities, a finding supported also by mutagenic analysis. Furthermore, inhibition studies indicate that S. solfataricus TrxR, structurally similar to eubacterial counterparts, is functionally closer to eukaryal TrxR. S. solfataricus NOX is a homodimeric flavo-enzyme able to oxidise both NADH and NADPH, and belongs to the NOX family forming H2O2 as end product of the reaction. The lack of cysteines in its primary structure indicates that the electron transfer from NAD(P)H to molecular oxygen does not involve a disulphide bridge.
The results of this investigation indicate that Sulfolobus solfataricus possesses efficient enzyme systems for the protection against oxidative stress. In particular, SOD and TrxR from this archaeal source share similar functional and molecular properties with their mitochondrial counterparts, thus supporting the hypothesis that the Sulfolobus genus is the putative ancestor of animal mitochondria. Vice versa, the properties of S. solfataricus NOX confirm that this enzyme belongs to a protein family scarcely conserved during evolution; the data also suggest a possible involvement of this enzyme in the protection against oxygen toxicity
IDENTIFICATION OF CHEMICALLY DIVERSE CDC25B PHOSPHATASE INHIBITORS BY RECEPTOR-BASED VIRTUAL SCREENING
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