Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1

Abstract

The interactive effects of temperature and cosolvents on the kinetic and structural features of a carboxylesterase from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1 (Sso EST1) were examined. While dimethylformamide, acetonitrile, and dioxane were all found to be deleterious to enzyme function, dimethyl sulfoxide (DMSO) activated Sso EST1 to various extents. This was particularly true at 3.5% (v/v) DMSO, where k(cat) was 20-30% higher than at 1.2% DMSO, over the temperature range of 50-85 degrees C. DMSO compensated for thermal activation in some cases; for example, k(cat) at 60 degrees C in 3.5% DMSO was comparable to k(cat) at 85 degrees C in 1.2% DMSO. The relationship between DMSO activation and enzyme structural characteristics was also investigated. Nuclear magnetic resonance spectroscopy and circular dichroism showed no gross change in enzyme conformation with 3.5% DMSO between 50 and 80 degrees C. However, low levels of DMSO were shown to have a small yet significant change in enzyme conformation. This was evident through the reduction of Sso EST1's melting temperature and changes in the microenvironment of the enzyme's tyrosine and tryptophan residues at 3.5% versus 1.2% (v/v) solvent. Finally, activation parameter analysis based on kinetic data, at 1.2% and 3.5% DMSO, implied an increase in conformational flexibility with additional cosolvent. These results suggest the activating effect of DMSO was related to small changes in the enzyme's structure resulting in an increase in its conformational flexibility. Thus, in addition to their use for solubilizing hydrophobic substrates in water, cosolvents may also serve as activators in applications involving thermostable biocatalysts at sub-optimal temperatures.

The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes

“…It has been reported that purified microbial and metagenomic esterases exhibit different sensitivities to organic solvents and detergents (Elend et al 2006;Fu et al 2011;Jiang et al 2012;Kang et al 2011). For example, the activity of the EstA esterase from Arthrobacter nitroguajacolicus was slightly stimulated (10-20 %) by the addition of DMSO (30 %) or acetonitrile (30 %), whereas lower concentrations (10 %) of these solvents reduced (20 to 90 %) the activity of esterases from Sulfolobus solfataricus (EST1) and Archaeoglobus fulgidus (Est-AF) (Kim et al 2008;Schutte and Fetzner 2007;Sehgal et al 2002). Similarly, several metagenomic esterases have been reported to be inhibited, unaffected or stimulated by the addition of DMSO (10-30 %), whereas all these enzymes were sensitive to acetonitrile and detergents (Triton X-100 and Tween-20) (Elend et al 2006;Fu et al 2011;Jiang et al 2012;Kang et al 2011).…”

Acidophilic Tannase from Marine Aspergillus awamori BTMFW032

“…Enzyme-catalyzed reactions in organic solvents, and even in supercritical fluids and the gas phase, have found numerous potential applications, some of which have already been commercialized. DMSO was reported to have an activating effect related to small changes in the enzyme's structure, resulting in an increase in its conformational flexibility [40]. Thus, co-solvents may also serve as activators in applications, in addition to their use for solubilizing hydrophobic substrates in water.…”

Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases

“…1). This observation points to the heightened structural rigidity of hyperthermophilic enzymes, even within their functional temperature ranges [48]. All XIs are known to be active only in the presence of divalent cations.…”