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.

A novel fatty acid binding protein produced by teratocytes of the aphid parasitoid Aphidius ervi.

Aphidius ervi is an endophagous braconid, parasitoid of the pea aphid, Acyrthosiphon pisum. A. ervi teratocytes, deriving from the dissociation of the embryonic serosa, synthesize and release two major proteins into the host haemocoel. The gene of one of these proteins has been cloned and characterized. This gene codes for a 15.8 kDa protein belonging to the fatty acid binding protein (FABP) family, named Ae-FABP (A. ervi-FABP). It is abundantly present in the host haemolymph when the parasitoid larva attains its maximum growth rate. The recombinant Ae-FABP binds to fatty acids in vitro, showing a high affinity to C14-C18 saturated fatty acids and to oleic and arachidonic acid. The possible nutritional role for the parasitoid larva of Ae-FABP is discussed.

Enzymatic synthesis and hydrolysis of xylogluco-oligosaccharides using the first archaeal alpha-xylosidase from Sulfolobus solfataricus.

The first, recently identified, archaeal alpha-xylosidase from Sulfolobus solfataricus (XylS) shows high specificity for hydrolysis of isoprimeverose [alpha-D-xylopyranosyl-(1,6)-D-glucopyranose, (X)], the p-nitrophenyl-beta derivative of isoprimeverose, and xyloglucan oligosaccharides and has transxylosidic activity, forming, in a retaining mode, interesting alpha-xylosides. This article describes the synthesis of isoprimeverose, the disaccharidic repeating unit of xyloglucan, of the p-nitrophenyl-beta derivative of isoprimeverose, and of a trisaccharide based on isoprimeverose that is one of the trisaccharidic building blocks of xyloglucan. A substrate structure-activity relationship is recognized for both the hydrolysis and the synthesis reactions of XylS, it being a biocatalyst (i) active hydrolytically only on X-ending substrates liberating a xylose molecule and (ii) capable of transferring xylose only on the nonreducing end glucose of p-nitrophenyl-(PNP)-beta-D-cellobioside. The compounds synthesized by this enzyme are a starting point for enzymological studies of other new enzymes (i.e., xyloglucanases) for which suitable substrates are difficult to synthesize. This study also allows us to define the chemical characteristics of the xylose-transferring activity of this new archaeal enzyme, contributing to building up a library of different glycosidases with high specific selectivity for oligosaccharide synthesis.

Enzymatic synthesis of oligosaccharides by two glycosyl hydrolases of Sulfolobus solfataricus.

The importance of carbohydrates in a variety of biological functions is the reason that interest has recently increased in these compounds as possible components of therapeutic agents. Thus, the need for a technique allowing the easy synthesis of carbohydrates and glucoconjugates is an emerging challenge for chemists and biologists involved in this field. At present, enzymatic synthesis has resulted in the most promising approach for the production of complex oligosaccharides. In this respect, the enzymological characteristics of the catalysts, in term of regioselectivity, substrate specificity, and operational stability, are of fundamental importance to improve the yields of the process and to widen the repertoire of the available products. Here, two methods of oligosaccharide synthesis performed by a glycosynthase and by an alpha-xylosidase from the hyperthermophilic archaeon Sulfolobus solfataricus are briefly reviewed. The approaches used and the biodiversity of the catalysts together are key features for their possible utilization in the synthesis of oligosaccharides.

Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal beta-glycosidases.

S beta gly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of S beta gly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure-function relationships in these enzymes.

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.

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.

A novel thermophilic glycosynthase that effects branching glycosylation.

A novel thermophilic glycosynthase that effects branching glycosylation has been obtained by mutation of the nucleophile in the active site of the glycosidase from Sulfolobus solfataricus. Two methods for the use of this mutant are reported.

Restoration of the activity of active-site mutants of the hyperthermophilic beta-glycosidase from Sulfolobus solfataricus: dependence of the mechanism on the action of external nucleophiles.

The beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus hydrolyzes beta-glycosides following a retaining mechanism based upon the action of two amino acids: Glu387, which acts as the nucleophile of the reaction, and Glu206, which acts as the general acid/base catalyst. The activities of inactive mutants of the catalytic nucleophile Glu387Ala/Gly were restored by externally added nucleophiles. Sodium azide and sodium formate were used as external nucleophiles and the products of their reaction were characterized. Glu387Ala/Gly mutants were reactivated with 2, 4-DNP-beta-Glc substrate and the Glu387Gly mutant showed recovered activity, with the same nucleophiles, also on 2-NP-beta-Glc. The reaction catalyzed by the Glu387Gly mutant proceeded differently depending on the type of externally added nucleophile. Sodium azide restored the catalytic activity of the mutant by attacking the alpha-side of the anomeric carbon of the substrates, thereby yielding an inverting glycosidase. Sodium formate promoted the opposite behavior (retaining) in the mutant, producing 3-O-beta-linked disaccharide derivative of the substrates. A possible role of sodium formate as a biomimicking agent in replacing the natural nucleophile Glu387 is also discussed.

Molecular biology of hyperthermophilic Archaea.

The sequences of a number of archaeal genomes have recently been completed, and many more are expected shortly. Consequently, the research of Archaea in general and hyperthermophiles in particular has entered a new phase, with many exciting discoveries to be expected. The wealth of sequence information has already led, and will continue to lead to the identification of many enzymes with unique properties, some of which have potential for industrial applications. Subsequent functional genomics will help reveal fundamental matters such as details concerning the genetic, biochemical and physiological adaptation of extremophiles, and hence give insight into their genomic evolution, polypeptide structure-function relations, and metabolic regulation. In order to optimally exploit many unique features that are now emerging, the development of genetic systems for hyperthermophilic Archaea is an absolute requirement. Such systems would allow the application of this class of Archaea as so-called "cell factories": (i) expression of certain archaeal enzymes for which no suitable conventional (mesophilic bacterial or eukaryal) systems are available, (ii) selection for thermostable variants of potentially interesting enzymes from mesophilic origin, and (iii) the development of in vivo production systems by metabolic engineering. An overview is given of recent insight in the molecular biology of hyperthermophilic Archaea, as well as of a number of promising developments that should result in the generation of suitable genetic systems in the near future.

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.

Crystal structure of the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability.

Enzymes from hyperthermophilic organisms must operate at temperatures which rapidly denature proteins from mesophiles. The structural basis of this thermostability is still poorly understood. Towards a further understanding of hyperthermostability, we have determined the crystal structure of the beta-glycosidase (clan GH-1A, family 1) from the hyperthermophilic archaeon Sulfolobus solfataricus at 2.6 A resolution. The enzyme is a tetramer with subunit molecular mass at 60 kDa, and crystallises with half of the tetramer in the asymmetric unit. The structure is a (betaalpha)8 barrel, but with substantial elaborations between the beta-strands and alpha-helices in each repeat. The active site occurs at the centre of the top face of the barrel and is connected to the surface by a radial channel which becomes a blind-ended tunnel in the tetramer, and probably acts as the binding site for extended oligosaccharide substrates. Analysis of the structure reveals two features which differ significantly from mesophile proteins; (1) an unusually large proportion of surface ion-pairs involved in networks that cross-link sequentially separate structures on the protein surface, and (2) an unusually large number of solvent molecules buried in hydrophilic cavities between sequentially separate structures in the protein core. These factors suggest a model for hyperthermostability via resilience rather than rigidity.

Identification of two glutamic acid residues essential for catalysis in the beta-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus.

The Sulfolobus solfataricus, strain MT4, beta-glycosidase (Ss beta-gly) is a thermophilic member of glycohydrolase family 1. To identify active-site residues, glutamic acids 206 and 387 have been changed to isosteric glutamine by site-directed mutagenesis. Mutant proteins have been purified to homogeneity using the Schistosoma japonicum glutathione S-transferase (GST) fusion system. The proteolytic cleavage of the chimeric protein with thrombin was only obtainable after the introduction of a molecular spacer between the GST and the Ss beta-gly domains. The Glu387-->Gln mutant showed no detectable activity, as expected for the residue acting as the nucleophile of the reaction. The Glu206-->Gln mutant showed 10- and 60-fold reduced activities on aryl-galacto and aryl-glucosides, respectively, when compared with the wild type. Moreover, a significant Km decrease with p/o-nitrophenyl-beta-D-glucoside was observed. The residual activity of the Glu206-->Gln mutant lost the typical pH dependence shown by the wild type. These data suggest that Glu206 acts as the general acid/base catalyst in the hydrolysis reaction.

Industrial-scale production and rapid purification of an archaeal beta-glycosidase expressed in Saccharomyces cerevisiae.

The application of enzymes isolated from extreme thermophiles in biotechnological processes is hampered by their unconventional fermentation conditions. The expression, in mesophilic hosts, of genes encoding for thermophilic proteins enables these difficulties to be overcome and permits the production of enzymes in high yield by using conventional fermentation plants and an efficient enzyme purification utilizing heat precipitation of host proteins. The beta-glycosidase gene from Sulfolobus solfataricus, a thermoacidophilic archaeon growing at 87 degrees C and pH 3.5, has been cloned and expressed in Saccharomyces cerevisiae (baker's yeast). The fermentation of a S. cerevisiae strain on a 100-litre scale and the two-step purification of the expressed beta-glycosidase by cell autolysis and extracts thermal precipitation is described. This procedure, after 72 h of autolysis, gave a yield 56-fold higher with respect to that obtained with the beta-glycosidase from S. solfataricus.

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.

A gene encoding a putative membrane protein homologous to the major facilitator superfamily of transporters maps upstream of the beta-glycosidase gene in the archaeon Sulfolobus solfataricus.

We have identified a gene encoding a putative membrane protein homologous to the major facilitator superfamily, mapping upstream of the lacS gene in Sulfolobus solfataricus. Permeases from this family mediate secondary transport and are widely distributed among eubacteria and eukaryotes; the finding of an archaeal member suggests that this mechanism of transport evolved before the divergence of the three living domains. We also report a transcriptional mapping of the gene cluster.

Molecular biology of extremophiles.

The molecular biology of extremophiles has recently attracted much interest, both in terms of cell adaptation to extreme environmental conditions and the development of manipulative genetic techniques. Although molecular genetic techniques have been successfully applied to halophiles and methanogens, their use with hyperthermophiles is limited by the extreme growth conditions that these organisms require. Much information on the thermophilic Archaea, has been obtained by studying the key enzymes involved in fundamental cell processes, such as transcription and replication, and by the cloning, sequence comparison and heterologous expression of structural genes. The development of viral vectors and systems for transformation, mutant production and screening will permit increased genetic manipulation of these organisms.

Effect of cavity-creating mutations in the hydrophobic core of chymotrypsin inhibitor 2.

Hydrophobic residues in the core of a truncated form of chymotrypsin inhibitor 2 (CI2) have been mutated in order to measure their contribution to the stability of the protein. The free energy of unfolding of wild-type and mutants was measured by both guanidinium chloride-induced denaturation and differential scanning calorimetry. The two methods give results for the changes in free energy on mutation that agree to within 1% or 2%. The average change in the free energy of unfolding (+/- standard deviation) for an Ile-->Val mutation is 1.2 +/- 0.1 kcal mol-1, for a Val-->Ala mutation 3.4 +/- 1.5 kcal mol-1, and for either an Ile-->Ala or a Leu-->Ala mutation 3.6 +/- 0.6 kcal mol-1. This gives an average change in the free energy of unfolding for deleting one methylene group of 1.3 +/- 0.5 kcal mol-1. Two significant correlations were found between the change in the free energy of unfolding between wild-type and mutant, delta delta GU-F, and the environment of the mutated residue in the protein. The first is between delta delta GU-F and the difference in side-chain solvent-accessible area buried between wild-type and mutant (correlation coefficient = 0.81, 10 points). The second and slightly better correlation was found between delta delta GU-F and N, the number of methyl/methylene groups within a 6-A radius of the hydrophobic group deleted (correlation coefficient = 0.84, 10 points). The latter correlation is very similar to that found previously for barnase, suggesting that this relationship is general and applies to the hydrophobic cores of other globular proteins. The combined data for C12 and barnase clearly show a better correlation with N (correlation coefficient = 0.87, 30 points) than with the change in the solvent-accessible surface area (correlation coefficient = 0.82, 30 points). This indicates that the packing density around a particular residue is important in determining the contribution the residue makes to protein stability. In one case, Ile-->Val76, a mutation which deletes the C delta 1 methyl group of a buried side chain, a surprising result was obtained. This mutant was found to be more stable than wild-type by 0.2 +/- 0.1 kcal mol-1. We have solved and analyzed the crystal structure of this mutant and find that there are small movements of side chains in the core, the largest of which, 0.7 A, is a movement of the side chain that has been mutated.(ABSTRACT TRUNCATED AT 400 WORDS)