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

Abstract

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.

Residues at the Active Site of the Esterase 2 fromAlicyclobacillus acidocaldarius Involved in Substrate Specificity and Catalytic Activity at High Temperature

“…Polar residues are in cyan. (9,19). However, we did not observe any significant change in the thermostability of variants compared with the wild type (Fig.…”

“…Structural investigations on EST2 were undertaken to study its structure-function relationships in detail (13,19,20). Furthermore, esterases are enzymes of considerable industrial potential.…”

The Aes Protein and the Monomeric α-Galactosidase fromEscherichia coli Form a Non-covalent Complex

“…Recently, we reported the three-dimensional structures of EST2 from Alicyclobacillus acidocaldarius, a thermophilic eubacterium (20), and of the homologous enzyme AFEST, from the hyper-thermophilic archaeon Archaeoglobus fulgidus (21). We also reported some structural and thermodynamic studies on both enzymes (22)(23)(24). To perform a comparative analysis with a mesophilic member of the HSL family we started the cloning of aes from E. coli genomic DNA, the overexpression of the protein in E. coli, and its purification.…”

Structural study of carboxylesterase from hyperthermophilic bacteria Geobacillus stearothermophilus by molecular dynamics simulation

“…Enzymes and proteins isolated from thermophilic microorganisms exhibit a high stability in conditions usually used for denaturing proteins such as high temperature, ionic strength, extreme pH values, elevated concentration of detergents and chaotropic agents [1,2]. As a result they can be used as biocatalysts under harsh environmental conditions [3]. In addition to the potential industrial applications, proteins and enzymes that are stable and active over 100 • C represent good models to shed light on the molecular adaptation of life at high temperature [4,5].…”

“…MD study has also been employed to study the dynamics of lipase [15], xylanase [16] dihydrofolate reductase [17], ribonuclease H1 [18] at high temperatures. So far MD simulation studies of psychrophilic esterase from Pseudoalteromonas haloplanktis [19,20], hyperthermophilic esterase from Archaeoglobus fulgidus [3] and thermostable paranitrobenzyl esterase from Bacillus subtilis [21] have been reported. However, to date no molecular dynamics simulation study of any hyperthermophilic carboxylesterase has been reported in the literature.…”