Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Abstract

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

“…4 increase ∆G by an entropic destabilization of the unfolded state [18][19][20]. However, particularly introduced disulfide bonds may affect the native state as well since they might prevent unfolding by tethering the native protein structure [21].…”

“…The kinetics of thermally induced unfolding was assessed by limited proteolysis with thermolysin under conditions where the observable rate constant of proteolysis corresponds to k U [47]. As disulfide bonds should predominantly affect the unfolded state [15,18,56], marginal changes in k U were expected for the disulfide variants in comparison to RNase A. In contrast, according to the unfolding region postulate k U should account for the changes in thermal stability for the unfolding region variants (R10C/R33C-, M30C/N44C-, and V43C/R85C-RNase A).…”

Metal ions affect insulin-degrading enzyme activity

“…Metal ions have long been recognized to stabilize and activate enzymes [37,38]. For several proteins, binding of metal cofactors, such as calcium, magnesium, manganese, copper, zinc and iron in different oxidation states, has been shown to increase the thermal stabilities of the proteins [39,40] and to substantially influence their structure and activity [41][42][43].…”

Self-assembly of trehalose molecules on a lysozyme surface: the broken glass hypothesis

“…That protein rigidity is a prerequisite for protein thermostability is a working hypothesis used by Vieille and Zeikus. 22 Lysozyme folding and unfolding takes far longer than is currently possible for anyone to simulateif the necessary number of water molecules and trehalose molecules are included. 23,24 .…”