Effects of temperature and SDS on the structure of <i>β</i>-glycosidase from the thermophilic archaeon <i>Sulfolobus solfataricus</i>

D’Auria, Sabato; Barone, Roberto; Rossi, Mosè; Nucci, Roberto; Barone, G.; Fessas, Dimitrios; Bertoli, Enrico; Tanfani, Fabio

doi:10.1042/bj3230833

Cited by 61 publications

(70 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In these experiments (Fig. 5), the endothermic unfolding transition for both proteins was superimposed to a sharp exothermic transition, indicative of aggregation phenomena (27). All the DSC transitions were irreversible under our conditions.…”

Section: Thermal Stability Studiesmentioning

confidence: 95%

Dissecting the Structural Determinants of the Stability of Cholesterol Oxidase Containing Covalently Bound Flavin

Caldinelli

Iametti

Barbiroli

et al. 2005

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His 69 residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (ϳ3 versus ϳ5 M, respectively), and the mutant protein had a melting temperature ϳ10 -15°C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 M urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.Bacterial cholesterol oxidase (CO, EC 1.1.3.6) is a monomeric bifunctional FAD-containing flavoenzyme that catalyzes the oxidation of 3␤-hydroxysteroids and the isomerization of the intermediate, ⌬ 5-6 -ene-3␤-ketosteroid (cholest-5-en-3-one) to produce ⌬ 3-4 -ene-3␤-ketosteroid (cholest-4-en-3-one). Bacteria that produce CO can be classified into two classes: non-pathogenic (e.g. Streptomyces and fast-growing Mycobacteria; these bacteria utilize cholesterol as their carbon source) and pathogenic bacteria (e.g. Rhodococcus equi and slow-growing Mycobacteria). Pathogenic bacteria require CO for infection of the host macrophage probably because of its ability to alter the physical structure of the membrane by converting cholesterol into cholest-4-en-3-one.The crystal structure of COs belonging to two distinct structural classes (1) have been solved by the Vrielink laboratory (2-4). Members classified as type I, such as those from Streptomyces sp. SA-COO and R. equi (5) have their cofactor tightly but non-covalently bound to the enzyme. A second CO from Brevibacterium sterolicum classified as type II, is completely different. In this enzyme the cofactor is covalently linked to the protein via a bond between the 8-methyl group of the isoalloxazine ring and the ND1 atom of His 69 .Although COs of class I an...

show abstract

Section: Thermal Stability Studiesmentioning

confidence: 95%

Dissecting the Structural Determinants of the Stability of Cholesterol Oxidase Containing Covalently Bound Flavin

Caldinelli

Iametti

Barbiroli

et al. 2005

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…At 98°C the band close to 1618 cm Ϫ1 reflects protein intermolecular interactions (aggregation) brought on by the thermal denaturation. 25 Keeping the protein at 98°C, for the time indicated in the Figure 3B (and Fig. 3 legend), results in a further protein denaturation as revealed by the decrease in intensity of the ␣-helix and ␤-sheet bands and by the increase in intensity of the band close to 1617 cm Ϫ1 due to aggregation.…”

Section: Thermal Denaturationmentioning

confidence: 97%

The thermophilic esterase fromArchaeoglobus fulgidus: Structure and conformational dynamics at high temperature

et al. 2000

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…CD spectra in the far-UV region and infrared spectroscopy at pH 10.0 showed negligible effects of SDS on the secondary structure of the enzyme, whereas more significant changes were observed in the protein tertiary structure by near-UV CD analysis (13).…”

mentioning

confidence: 99%

SDS-resistant Active and Thermostable Dimers Are Obtained from the Dissociation of Homotetrameric β-Glycosidase from Hyperthermophilic Sulfolobus solfataricus in SDS

Gentile

Amodeo

Febbraio

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

␤-Glycosidases are fundamental, widely conserved enzymes. Those from hyperthermophiles exhibit unusual stabilities toward various perturbants. Previous work with homotetrameric ␤-glycosidase from hyperthermophilic Sulfolobus solfataricus (M r 226,760) has shown that addition of 0.05-0.1% SDS was associated with minimal secondary structure perturbations and increased activity. This work addresses the effects of SDS on ␤-glycosidase quaternary structure. In 0.1-1% SDS, the enzyme was dimeric, as determined by Ferguson analysis of transverse-gradient polyacrylamide gels. The catalytic activity of the ␤-glycosidase dimer in SDS was determined by in-gel assay. A minor decrease of thermal stability in SDS was observed after exposure to temperatures up to 80°C for 1 h. An analysis of ␤-glycosidase crystal structure showed different changes in solventaccessible surface area on going from the tetramer to the two possible dimers (A-C and A-D). Energy minimization and molecular dynamics calculations showed that the A-C dimer, exhibiting the lowest exposed surface area, was more stabilized by a network of polar interactions. The charge distribution around the A-C interface was characterized by a local short range anisotropy, resulting in an unfavorable interaction with SDS. This paper provides a detailed description of an SDS-resistant inter-monomeric interface, which may help understand similar interfaces involved in important biological processes.Enzymes from hyperthermophiles are interesting for the possible biotechnological applications of their unusual stability toward a number of perturbing agents, including extremes of temperature, pH, ionic strength, and detergents. In particular, the structural determinants of protein resistance to denaturation and dissociation by SDS have recently become the focus of increasing attention, as SDS-resistant intermolecular interactions have been identified within the context of important biological functions. A correlation was found between the SDS stability of different heterodimeric products of the major histocompatibility complex class II polymorphic genes of the HLA complex and susceptibility to important human autoimmune diseases, such as insulin-dependent diabetes mellitus (IDDM) 1 (1). Moreover, the isoform-specific formation of SDS-stable complexes was observed between amyloid-␤ (A␤), a major component of extracellular senile plaques of Alzheimer's disease (AD), and the apolipoproteins apoE2 and apoE3 but not with apoE4, which is present with increased frequency in patients with sporadic and late-onset familial AD, and is considered a risk factor for the disease (2, 3). Investigations of the mechanistic basis of these SDS-stable interactions have pinpointed some of the critical amino acid residues and the interactions involved (4 -7).Proteins from hyperthermophilic organisms are ideally suited for the study of the structural determinants of protein stability, in view of their physicochemical resistance, higher phylogenetic proximity to eukaryotic enzymes, in comparison with p...

show abstract

Effects of temperature and SDS on the structure of β-glycosidase from the thermophilic archaeon Sulfolobus solfataricus

Cited by 61 publications

References 39 publications

Dissecting the Structural Determinants of the Stability of Cholesterol Oxidase Containing Covalently Bound Flavin

Dissecting the Structural Determinants of the Stability of Cholesterol Oxidase Containing Covalently Bound Flavin

The thermophilic esterase fromArchaeoglobus fulgidus: Structure and conformational dynamics at high temperature

SDS-resistant Active and Thermostable Dimers Are Obtained from the Dissociation of Homotetrameric β-Glycosidase from Hyperthermophilic Sulfolobus solfataricus in SDS

Contact Info

Product

Resources

About