beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: structure and activity in the presence of alcohols.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a tetrameric protein with a molecular mass of 240 kDa, stable in the presence of detergents, and with a maximal activity at temperatures above 95 degrees C. Understanding the structure-activity relationships of the enzyme under different conditions is of fundamental importance for both theoretical and applicative purposes. In this paper we report the effect of methanol, ethanol, 1-propanol, and 1-butanol on the activity of S. solfataricus beta-glycosidase expressed in Escherichia coli. The alcohols stimulated the enzyme activity, with 1-butanol producing its maximum effect at a lower concentration than the other alcohols. The structure of the enzyme was studied in the presence of 1-butanol by circular dichroism, and Fourier-transform infrared and fluorescence spectroscopies. Circular dichroism and steady-state fluorescence measurements revealed that at low temperatures the presence of the alcohol produced no significant changes in the tertiary structure of the enzyme. However, time-resolved fluorescence data showed that the alcohol modifies the protein microenvironment, leading to a more flexible enzyme structure, which is probably responsible for the enhanced enzymatic activity.     

Structural basis of the destabilization produced by an amino-terminal tag in the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus

We have previously shown that the major ion-pairs network of the tetrameric beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus involves more than 16 ion-pairs and hydrogen bonds between several residues from the four subunits and protects the protein from thermal unfolding by sewing the carboxy-termini of the enzyme. We show here that the amino-terminal of the enzyme also plays a relevant role in the thermostabilization of the protein. In fact, the addition of four extra amino acids at the amino-terminal of the beta-glycosidase, though not affecting the catalytic machinery of the enzyme and its thermophilicity, produced a faster enzyme inactivation in the temperature range 85-95 degrees C and decreased the Tm of the protein of 6 degrees C, measured by infrared spectroscopy. In addition, detailed two-dimensional IR correlation analysis revealed that the quaternary structure of the tagged enzyme is destabilized at 85 degrees C whilst that of the wild type enzyme is stable up to 98 degrees C. Molecular models allowed the rationalization of the experimental data indicating that the longer amino-terminal tail may destabilize the beta-glycosidase by enhancing the molecular fraying of the polypeptide and loosening the dimeric interfaces. The data support the hypothesis that fraying of the polypeptide chain termini is a relevant event in protein unfolding.     

Identification and molecular characterization of the first alpha -xylosidase from an archaeon

We here report the first molecular characterization of an alpha-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 degrees C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to alpha-glucosidases, alpha-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a beta-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The alpha-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90 degrees C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the alpha-xylosidase and the beta-glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.     

The thermophilic esterase from Archaeoglobus fulgidus: structure and conformational dynamics at high temperature

The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa. The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C. We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The data show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of beta-sheets. This finding may be surprising for a thermophilic protein and suggests the presence of a temperature-sensitive beta-sheet. The increase in temperature from 60 degrees C to 98 degrees C induced a decrease of alpha-helix and beta-sheet bands which, however, are still easily detected at 98 degrees C indicating that at this temperature some secondary structure elements of the protein remain intact. The conformational dynamics of the esterase were investigated by frequency-domain fluorometry and anisotropy decays. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80 degrees C, as shown from the emission spectra and by acrylamide quenching experiments. The relationship between enzyme activity and protein structure is discussed.     

The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro.

We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 degrees C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium-dependent ATPase activity with an optimum temperature at 80 degrees C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride-denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible inactivations at 90 degrees C of several thermophilic enzymes by the binding of the denaturation intermediate; the time-courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 degrees C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.     

Thermal stability and aggregation of sulfolobus solfataricus beta-glycosidase are dependent upon the N-epsilon-methylation of specific lysyl residues: critical role of in vivo post-translational modifications

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of beta-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native beta-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of beta-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of beta-glycosidase from S. solfataricus.     

Interaction of the ADP-ribosylating enzyme from the hyperthermophilic archaeon S. solfataricus with DNA and ss-oligo deoxy ribonucleotides

The DNA-binding ability of the poly-ADPribose polymerase-like enzyme from the extremely thermophilic archaeon Sulfolobus solfataricus was determined in the presence of genomic DNA or single stranded oligodeoxyribonucleotides. The thermozyme protected homologous DNA against thermal denaturation by lowering the amount of melted DNA and increasing melting temperature. The archaeal protein induced structural changes of the nucleic acid by modifying the dichroic spectra towards a shape typical of condensing DNA. However, enzyme activity was slightly increased by DNA. Competition assays demonstrated that the protein interacted also with heterologous DNA. In order to characterize further the DNA binding properties of the archaeal enzyme, various ss-oligodeoxyribonucleotides of different base composition, lengths (12-mer to 24-mer) and structure (linear and circular) were used for fluorescence titration measurements. Intrinsic fluorescence of the archaeal protein due to tryptophan (excitation at 295 nm) was measured in the presence of each oligomer at 60 degrees C. Changes of tryptophan fluorescence were induced by all compounds in the same range of base number per enzyme molecule, but independently from the structural features of oligonucleotides, although the protein exhibited a slight preference for those adenine-rich and circular. The binding affinities were comparable for all oligomers, with intrinsic association constants of the same order of magnitude (K=10(6) M(-1)) in 0.01 M Na-phosphate buffer, pH 8.0, and accounted for a "non-specific" binding protein. Circular dichroism analysis showed that at 60 degrees C the native protein was better organized in a secondary structure than at 20 degrees C. Upon addition of oligonucleotides, enzyme structure was further stabilized and changed towards a beta-conformation. This effect was more marked with the circular oligomer. The analysed oligodeoxyribonucleotides slightly enhanced enzyme activity with the maximal increase of 50% as compared to the control. No activation was observed with the circular oligomer.     

Sensitive monitoring of the dynamics of a membrane-bound transport protein by tryptophan phosphorescence spectroscopy

This paper presents a tryptophan phosphorescence spectroscopy study on the membrane-bound mannitol transporter, EII(mtl), from E. coli. The protein contains four tryptophans at positions 30, 42, 109, and 117. Phosphorescence decays in buffer at 1 degrees C revealed large variations of the triplet lifetimes of the wild-type protein and four single-tryptophan-containing mutants. They ranged from <70 microseconds for the tryptophan at position 109 to 55 ms for the residue at position 30, attesting to widely different flexibilities of the tryptophan microenvironments. The decay of all tryptophans is multiexponential, reflecting multiple stable conformations of the protein. Both mannitol binding and enzyme phosphorylation had large effects on the triplet lifetimes. Mannitol binding induces a more ordered structure near the mannitol binding site, and the decay becomes significantly more homogeneous. In contrast, enzyme phosphorylation induces a large relaxation of the protein structure at the reporter sites. The implications of these structural changes on the coupling mechanism between the transport and the phosphorylation activity of EII(mtl) are discussed. Taken as a whole, our data show that tryptophan phosphorescence spectroscopy is a very sensitive technique to explore conformational dynamics in membrane proteins.     

Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus.

3-Phosphoglycerate kinases from yeast and the extreme thermophilic bacterium Thermus thermophilus HB8 have been used as models for investigating the relationship between stability, dynamics and activity. It was found that while at a given temperature the thermophilic protein is more stable, its conformational dynamics as measured by the ability of acrylamide to quench the fluorescence of a buried tryptophan as well as its specific activity, are both lower than for the mesophilic protein. As the temperature is increased, the thermodynamic stability of the thermophilic protein approaches that of the mesophilic protein at its working temperature. Its conformational dynamics and specific activity however were both shown to increase, until at the physiologically operational temperature, they become similar to those of the mesophilic enzyme at its operational temperature. These results confirm the proposal that a direct relationship and balance holds between thermodynamic stability, dynamics and specific activity in globular proteins. They demonstrate also the constraining effect of increased stability upon conformational dynamics and enzyme activity.     

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

A spectrofluorometric study of the environment of tryptophans in bacteriorhodopsin

The emission spectrum of intact purple membranes of Halobacterium halobium has a very short wavelength position (the main maximum at 314 nm) and can be fitted by two spectral components, one of which (component A) corresponds to the fluorescence of buried tryptophan residues located in a highly hydrophobic rigid environment (like the single tryptophan residue in azurin), the other (component I) being due to the emission of buried tryptophan residues located in a rather polar environment. Treatment of bacteriorhodopsin by NaBH4, fragmentation of the membranes and thermal formation of vesicles result in a decrease in the contribution of component A, an increase in that of component I and the appearance of spectral components corresponding to the emission of surface tryptophan residues. Temperature induces at least two distinct changes of the fluorescence parameters of the protein: one change occurs from 45 to 65 degrees C. the other from 65 to 90 degrees C. The spectral changes correlate with the peaks of heat sorption caused by thermal transitions in the purple membrane structure and conformational changes in the protein structure. Alkaline denaturation of bacteriorhodopsin registered by tryptophan fluorescence begins at pH > 11.0.     

Conformational studies of a hyperthermostable enzyme

The structural features of the hyperthermophilic endo-beta-1,3-glucanase from Pyrococcus furiosus were studied using circular dichroism, steady-state and time-resolved fluorescence spectroscopy and anisotropy. Upon heat and chemical treatment the folded and denatured states of the protein were characterized by distinguishable spectral profiles that identified a number of conformational states. The fluorescence methods showed that the spectral differences arose from changes in the local environment around specific tryptophan residues in the native, partially folded, partially unfolded and completely unfolded state. A structural resemblance was observed between the native protein and the structurally perturbed state which resulted after heat treatment at 110 degrees C. The enzyme underwent disruption of the native secondary and tertiary structure only after incubation at biologically extremely high temperatures (i.e. 150 degrees C), whilst in the presence of 8 m of guanidine hydrochloride the protein was partially unfolded.     

Effect of temperature on intramolecular dynamics and conformational state of bacterial alkaline phosphatase

The slow internal dynamics and the conformational state of Escherichia coli alkaline phosphatase by the action of temperature in the range 0-100 degrees C have been investigated by tryptophan room temperature phosphorescence and fluorescence. It has been shown that heating an alkaline phosphatase solution in the interval 0-70 degrees C leads to a substantial increase in the slow internal dynamics. A further increase in temperature to 95 degrees C causes a reversible enhancement of internal dynamics and a partial unfolding of the globule. Heating the protein solution in a narrow temperature range 97-100 degrees C induces an irreversible conformational transition, which is characterized by total unfolding of the globule, a drastic increase in internal dynamics, and the loss of enzymatic activity.     

Effect of temperature on tryptophan fluorescence of beta-lactoglobulin B.

The effect of heat on the conformation of bovine beta-lactoglobulin has been studied using intrinsic fluorescence spectroscopy. Changes in the intensity, wave-length of maximum emission and emission peak width at half height of tryptophan fluorescence over the range 15-90 degrees C at pH 6.4-6.5 has allowed the environments of the two tryptophans in the molecule to be discriminated. At 20 degrees C both tryptophans are in hydrophobic environments. As the temperature is raised the conformation changes such that at about 50 degrees C one of the tryptophans is transferred to a more polar environment accessible to solvent. Conformational changes appear to be reversible if the protein is cooled to 20 degrees C after heat treatments up to 70 degrees C. Above 70 degrees C the second tryptophan residue becomes exposed to solvent. Complete exposure of one residue occurs at 80 degrees C while the other is still partially buried even at 90 degrees C. When the protein is then cooled to 20 degrees C the conformational changes appear to be irreversible with only one tryptophan residue returning to the hydrophobic interior of the molecule.     

Thermostable lipase of Bacillus Stearothermophilus: high-level production, purification, and calcium-dependent thermostability

An efficient expression system was developed for the production of the thermostable lipase from Bacillus stearothermophilus L1 in an Escherichia coli system. A structural gene corresponding to mature lipase was subcloned in the pET-22b(+) expression vector and its expression was induced by IPTG at 30 degrees C in E. coli cells. The lipase activity in a cell-free extract was as high as 448,000 units/g protein, which corresponds to as much as 26% of the total cellular protein and is 77 times higher than that of E. coli RR1/pLIP1. Based on its pI (7.4) and pH stability data reported previously, the L1 lipase was efficiently purified to homogeneity with CM (at pH 6.0) and DEAE (at pH 8.8) column chromatographies with a recovery yield of 62%. The specific activity of the purified enzyme was 1700 units/mg protein when olive oil emulsion was used as a substrate. Its optimum temperature for the hydrolysis of olive oil was 68 degrees C and it was stable up to 55 degrees C for 30 min-incubation. The thermostability increased by about 8-10 degrees in the presence of calcium ions. This calcium-dependent thermostability was confirmed by the tryptophan fluorescence emission kinetics showing that the enzyme starts to unfold at 66 degrees C in the presence of calcium ions but at 58 degrees C in the absence of calcium ions, implying that the calcium ions bind to the thermostable enzyme and stabilize the protein tertiary structure even at such high temperatures.     

The chromosomal protein sso7d of the crenarchaeon Sulfolobus solfataricus rescues aggregated proteins in an ATP hydrolysis-dependent manner

In this work, we show that the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus displays a cation-dependent ATPase activity with a pH optimum around neutrality and a temperature optimum of 70 degrees C. Measurements of tryptophan fluorescence and experiments that used 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that ATP hydrolysis induces a conformational change in the molecule and that the binding of the nucleotide triggers the ATP hydrolysis-induced conformation of the protein to return to the native conformation. We found that Sso7d rescues previously aggregated proteins in an ATP hydrolysis-dependent manner; the native conformation of Sso7d forms a complex with the aggregates, while the ATP hydrolysis-induced conformation is incapable of this interaction. Sso7d is believed to be the first protein isolated from an archaeon capable of rescuing aggregates.     

The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.     

Effect of molecular confinement on internal enzyme dynamics: frequency domain fluorometry and molecular dynamics simulation studies

The tryptophanyl emission decay of the mesophilic beta-galactosidase from Aspergillus oryzae free in buffer and entrapped in agarose gel is investigated as a function of temperature and compared to that of the hyperthermophilic enzyme from Sulfolobus solfataricus. Both enzymes are tetrameric proteins with a large number of tryptophanyl residues, so the fluorescence emission can provide information on the conformational dynamics of the overall protein structure rather than that of the local environment. The tryptophanyl emission decays are best fitted by bimodal Lorentzian distributions. The long-lived component is ascribed to close, deeply buried tryptophanyl residues with reduced mobility; the short-lived one arises from tryptophanyl residues located in more flexible external regions of each subunit, some of which are involved in forming the catalytic site. The center of both lifetime distribution components at each temperature increases when going from the free in solution mesophilic enzyme to the gel-entrapped and hyperthermophilic enzyme, thus indicating that confinement of the mesophilic enzyme in the agarose gel limits the freedom of the polypeptide chain. A more complex dependence is observed for the distribution widths. Computer modeling techniques are used to recognize that the catalytic sites are similar for the mesophilic and hyperthermophilic beta-galactosidases. The effect due to gel entrapment is considered in dynamic simulations by imposing harmonic restraints to solvent-exposed atoms of the protein with the exclusion of those around the active site. The temperature dependence of the tryptophanyl fluorescence emission decay and the dynamic simulation confirm that more rigid structures, as in the case of the immobilized and/or hyperthermophilic enzyme, require higher temperatures to achieve the requisite conformational dynamics for an effective catalytic action and strongly suggest a link between conformational rigidity and enhanced thermal stability.     

Studies on the temperature effect on bacteriorhodopsin of purple and blue membrane by fluorescence and absorption spectroscopy

Fluorescence and absorption spectra were used to study the temperature effect on theconformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRprespectively).The maximum emission wavelengths of tryptophan fluorescence in both proteins at roomtemperature are 340 nm,and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of bRp.As temperature increases,the tryptophan fluorescence of bRb decreases,while the tryptophan fluorescenceof bRp increases.The binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bindonly to bRb,but not to bRp.These results suggest that significant structural difference existed between bRband bRp.It was also found that both kinds of bR are highly thermal stable.The maximum wavelength of theprotein fluorescence emission only shifted from 340 nm to 346 nm at 100℃.More interestingly,as tempera-ture increased,the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at380 nm formed.The transition occurred at a narrow temperature range (65℃-70℃).These facts indicatedthat an intermediate can be induced by high temperature.This phenomenon has not been reported before.