Antioxidant activity of the main bioactive derivatives from oleuropein hydrolysis by hyperthermophilic beta-glycosidase.

The main reaction products obtainable by the hydrolysis of commercially available oleuropein by hyperthermophilic beta-glycosidase were purified and structurally characterized by UV and 1H and 13C NMR analyses. Their antioxidant activity, in particular their capacity to inhibit the fatty acid peroxidation rate, was studied. The molecular structures assigned revealed the presence of two elenolic acid forms presenting different antioxidant abilities closely correlated to their molecular structures, as well as an unstable elenolate which is a rearrangement product of the oleuropein aglycon. This molecule, under the reaction conditions (pH 7.0, 60 degrees C) required for beta-glycosidase activity, rapidly gives rise to 3,4-dihydroxy-phenylethanol (hydroxytyrosol).

Intramolecular dynamics and conformational transition in proteins studied by biophysical labelling methods. Common and specific features of proteins from thermophylic micro-organisms.

A general survey is carried out on the theoretical grounds for methods of spin, luminescence and Mössbauer labels, as well as their application in the study of protein intramolecular dynamics. When combined, these methods allow the protein dynamics to be investigated within a wide range of correlation times (tau c = 10(2) - 10(-10) s) and amplitudes. The purposeful application of the methods to various proteins at different temperatures (30-330 K), water content, substrate addition, etc., revealed a number of dynamical processes and conformational transitions in proteins. The experiments indicated correlations between the local segmental mobility of protein globules in a nanosecond temporal scale and biochemical reactions, such as long-distance electron transfer, hydrolysis and photoreactions. The biophysical labelling methods results were analysed together with the data on dynamics obtained using complementary physico-chemical methods and theoretical calculations. Special emphasis is given to recent results on proteins from thermophylic micro-organisms. The mechanisms of protein intramolecular dynamics and their role in the stability and functions of proteins and enzymes are discussed.

Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus.

The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.

Hydrolysis of oleuropein by recombinant beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus immobilised on chitosan matrix.

The recombinant beta-glycosidase (EcS beta gly) from Sulfolobus solfataricus was immobilised on chitosan to perform the enzymatic hydrolysis of commercial oleuropein (heterosidic ester of elenolic acid and 3,4-dihydroxy-phenylethanol (hydroxytyrosol)) at two temperatures (60 and 70 degrees C). Interestingly, on the basis of the reasonable assumption that the enzyme hydrolyses only the sugar linkage, the biotransformation produces unstable aglycone species formed by oleuropein hydrolysis that, differently from some commercially available beta-glucosidases tested, give rise to the formation of hydroxytyrosol, at the operative temperatures of the bioreactor. The results of the biotransformation at 70 degrees C showed that the main products are hydroxytyrosol, and glucose, being the oleuropein aglycone present in low amount at the end of reaction. Both in single step approach or in recycle approach the amounts of glucose and oleuropein aglycone were lightly dependent from flow rate. The amount of hydroxytyrosol, increased on decreasing the flow rate of bioreactor in recycle approach, following a non-linear trend and obtaining the highest value at a flow rate of 15 ml h-1 while in the single step approach the 3,4-dihydroxy-phenylethanol was at its maximum at higher flow rate (16 ml h-1). For the hydrolysis of the oleuropein by bioreactor at 60 degrees C we used lower molar ratio oleuropein/enzyme only by the single step approach. In these conditions it is possible to obtain high amounts of only two products (glucose and hydroxytyrosol) in short time (2 h). The stability of the bioreactor at the operative temperatures showed a t1/2 of 30 days at 70 degrees C and a t1/2 of 56 days at 60 degrees C.

Purification and characterization of a lipoxygenase enzyme from durum wheat semolina.

Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 +/- 5 kDa. Among the substrates tested, linoleic acid showed the highest k(cat)/K(m) value; a beta-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6. 8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.

Homology modeling and active-site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2.

The moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structures predictions and a secondary structure-driven multiple sequence alignment with remote homologous protein of known three-dimensional (3D) structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the catalytic triad. In this paper we report the construction of a 3D model for this enzyme based on the structure of mouse acetylcholinesterase complexed with fasciculin. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser155, Asp252, and His282 are located close to each other at hydrogen bond distances. Their catalytic role was here probed by biochemical and mutagenic studies. Moreover, on the basis of the secondary structure-driven multiple sequence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzyme was drastically reduced. We propose that Gly84 is part of a putative "oxyanion hole" involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.

Structure-function studies on beta-glycosidase from Sulfolobus solfataricus. Molecular bases of thermostability.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a thermostable tetrameric protein with a molecular mass of 240 kDa which is stable in the presence of detergents and has a maximal activity above 95 degrees C. An understanding of the structure-function relationship of the enzyme under different chemical-physical conditions is of fundamental importance for both theoretical and application purposes. In this paper we report the effect of basic pH values on the structural stability of this enzyme. The structure of the enzyme was studied at pH 10 and in the temperature range 25-97.5 degrees C using circular dichroism, Fourier-transform infrared and fluorescence spectroscopy. The spectroscopic data indicated that the enzyme stability was strongly affected by pH 10 suggesting that the destabilization of the protein structure is correlated with the perturbation of ionic interactions present in the native protein at neutral pHs. These experiments give support to the observation derived from the 3D-structure, that large ion pair networks on the surface stabilize Sulfolobus solfataricus beta-glycosidase.

Advantages in using immobilized thermophilic beta-glycosidase in nonisothermal bioreactors.

Catalytic membranes, obtained by immobilizing thermophilic beta-glycosidase onto nylon supports, were used in a nonisothermal bioreactor to study the effect of temperature gradients on the rate of enzyme reaction. Two experimental approaches were carried out to explain the molecular mechanisms by which the temperature gradients affect enzyme activity. The results showed that the thermophilic enzyme behaved as the mesophilic beta-galactosidase, exhibiting an activity increase which was linearly proportional to the transmembrane temperature difference. The efficiency of the system proposed was determined by calculating two constants, alpha and beta, which represent respectively the percentage increase of enzyme activity when a temperature difference of 1 degrees C or a temperature gradient of 1 degrees C cm-1 were applied across the catalytic membrane. The increase of enzyme activity in nonisothermal bioreactors entailed a proportional reduction of production times. The advantages in using thermophilic enzymes immobilized in nonisothermal bioreactors are also discussed.

Identification of the active site nucleophile in the thermostable beta-glycosidase from the archaeon Sulfolobus solfataricus expressed in Escherichia coli.

Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli was fully inactivated at 65 degrees C, according to pseudo-first-order kinetics, by [3H]conduritol B epoxide (DL-1,2 anhydro-myo-inositol) synthesized as the active site directed inhibitor by a slight modification of Legler's procedure [Legler, G. (1977) Methods Enzymol. 46, 368-381]. The determination of kinetic constants for the inactivation showed that the process took place through the formation of a stabilized inhibitor-enzyme intermediate. Inactivation and reactivation studies suggested that the inhibitor-enzyme intermediate complex was formed more rapidly and hydrolyzed at a lower rate than it was for other glycosidases. Moreover, the stoichiometry of the binding, determined by electrospray mass spectrometric analysis, revealed that one molecule of the inhibitor was covalently bound to each enzyme subunit. The binding site for [3H]conduritol B epoxide was identified by the isolation and partial sequence analysis of the radioactive peptide obtained by cyanogen bromide and pepsin digests. Electrospray tandem mass analysis of the labeled peptide showed that the inhibitor was covalently bound to E387. This result, in agreement with data obtained from sequence alignments of S. solfataricus beta-glycosidase with other gluco- and galactosidases of the glycosyl hydrolase family 1 [Henrissat, B. (1991) Biochem. J. 280, 309-316], indicates that the conserved E387 is the nucleophilic amino acid residue in the active site of the enzyme.

Functional and structural properties of the homogeneous beta-glycosidase from the extreme thermoacidophilic archaeon sulfolobus solfataricus expressed in Saccharomyces cerevisiae.

A protein with beta-glycosidase activity from Sulfolobus solfataricus (S beta gly) was expressed in the yeast Saccharomyces cerevisiae. The purification procedure was made fast and easy by employing a single chromatographic step. After 5.8-fold purification, the cell extract gave a homogeneous enzyme at 166 U/mg. The recombinant enzyme was functionally and structurally similar to the wild-type enzyme. Kinetic experiments showed the same wide substrate specificity; in fact, the expressed enzyme hydrolyzed beta-D-gluco-, fuco-, and galactosides and a large number of glucoside dimers and oligomers, linked beta 1 -> 4. Moreover, the molecular mass of the enzyme was estimated to be 60 kDa by SDS-PAGE and 240 kDa by gel filtration, glycerol gradient, and ultracentrifugation analyses, indicating that the enzyme has a tetrameric structure. The N-terminal amino acid sequence, the temperature dependent activity, and content of secondary structure were similar to those of the wild-type enzyme. CD spectral and kinetic analyses showed that the only differences from the wild-type enzyme consist of the absence of lysine methylation, the presence of some glycosylated amino acid residues, and lower thermostability. Furthermore, calorimetric analyses on the expressed protein indicated values of delta dH = 5072 kJ/ mol and delta (d)C(p)= 100 kJ/mol, appreciably lower than those of the wild-type protein.

Expression and extensive characterization of a beta-glycosidase from the extreme thermoacidophilic archaeon Sulfolobus solfataricus in Escherichia coli: authenticity of the recombinant enzyme.

The gene coding for the beta-glycosidase from the archaeon Sulfolobus solfataricus has been overexpressed in Escherichia coli. The enzyme was purified to homogeneity with a rapid purification procedure employing a thermal precipitation as a crucial step. The final yield was 64% and the purification from the thermal precipitation was 5.4-fold. The expressed enzyme shows the same molecular mass, thermophilicity, thermal stability, and broad substrate specificity, with noticeable exocellobiase (glucan 1,4-beta-D-glucosidase) activity, of the enzyme purified from S. Solfataricus. We provide evidence that the beta-glycosidase can assume its functional state in E. coli without the contribution of N-epsilon-methylated lysine residues.

Interaction of the high-affinity inhibitor tetrahydro-dUMP with the allosteric enzyme deoxycytidylate aminohydrolase.

Tetrahydro-dUMP, an analog of the putative transition state in aminohydrolysis of deoxycytidine monophosphate (dCMP) inhibits the allosteric enzyme deoxycytidylate aminohydrolase with high affinity. The inhibition is reversible, and its kinetics is consistent with the analog binding at the substrate site only to one and the same conformation that binds the substrate dCMP. Such kinetics is what would be expected for a transition state analog interacting in an allosteric "K system."