Ion Pairs Involved in Maintaining a Thermostable Structure of Glutamate Dehydrogenase from a Hyperthermophilic Archaeon

Rahman, Raja Noor Zaliha Abd.; Fujiwara, Shinsuke; Nakamura, Haruki; Takagi, Masahiro; Imanaka, Tadayuki

doi:10.1006/bbrc.1998.8933

Cited by 50 publications

(33 citation statements)

References 27 publications

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“…Vetriani and coworkers (36) successfully improved the thermostability of a glutamate dehydrogenase from Thermococcus litoralis by introducing an ion pair network that matches the structure of the more thermostable P. furiosus glutamate dehydrogenase. Similar results have been described for glutamate dehydrogenase from T. kodakarensis KOD1 (37). Recently, introduction of a single mutation (Glu99 ¡ Gln) into the Pyrococcus horikoshii CutA1 protein was found to lead to an increase in the denaturation temperature of the protein, and the E99Q mutant was stabilized by deletion of the negatively charged group at the C terminus of an ␣ helix, in addition to elimination of the repulsive interactions with the other two Glu residues (38).…”

Section: Discussionsupporting

confidence: 78%

Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca ²⁺ -Binding Site

Zeng

Gao

Dai

et al. 2014

Appl Environ Microbiol

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Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little is known about the effects of metal ions on the properties of pyrolysin. Here, we report that the supplementation of Na ؉ , Ca 2؉ , or Mg 2؉ salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin but leads to an increase in enzyme activity. The destabilizing effect of metal ions on pyrolysin appears to be related to the disturbance of surface electrostatic interactions of the enzyme. In addition, mutational analysis of two predicted high-affinity Ca 2؉ -binding sites (Ca1 and Ca2) revealed that the binding of Ca 2؉ is important for the stabilization of this enzyme. Interestingly, Asn substitutions at residues Asp818 and Asp820 of the Ca2 site, which is located in the C-terminal extension of pyrolysin, resulted in improvements in both enzyme thermostability and activity without affecting Ca 2؉ -binding affinity. These effects were most likely due to the elimination of unfavorable electrostatic repulsion at the Ca2 site. Together, these results suggest that metal ions play important roles in modulating the stability and activity of pyrolysin. Hyperthermophiles grow optimally at temperatures of 80°C or higher (1), and some can survive and reproduce at temperatures higher than 120°C (2, 3). Enzymes from hyperthermophiles are especially valuable for probing the stabilization mechanisms that allow proteins to function near the maximum temperature of life, and studying these enzymes also offers insight into opportunities to greatly expand the reaction conditions of biocatalysis (4-6). At high temperatures, inactivation of enzymes occurs mainly due to thermal denaturation (loss of tertiary structure) and degradation (loss of primary structure) (7). Accumulating evidence suggests that hyperthermophilic enzymes have evolved multiple mechanisms to adapt to hyperthermal environments; however, no general rules for stabilization of these enzymes have been described (5,8). Known intrinsic stabilization factors include improved hydrophobic and aromatic interactions, increased ionic interactions, decreased entropy of the unfolded state and solvent-accessible hydrophobic surface, anchoring of loose ends, intersubunit interactions and oligomerization, metal binding, and posttranslational modifications (5, 8-11). Certain hyperthermophiles possess a specific protein disulfide oxidoreductase and use disulfide bonding as a major mechanism for protein stabilization (12, 13). In addition to the intrinsic factors, some hyperthermophilic enzymes are stabilized by extrinsic factors, such as intracellular compatible solutes and molecular chaperones, ligand binding, crowding effects inside the cells, and association with the surface layer (S-layer) (5,8,14,15). Although hyperthermophilic enzymes have become model systems to investigate mechanisms of enzyme stabilization and have provided the rational basis for improving the stability ...

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Section: Discussionsupporting

confidence: 78%

Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca ²⁺ -Binding Site

Zeng

Gao

Dai

et al. 2014

Appl Environ Microbiol

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“…The same ion pair network was created in P. kodakaraensis GDH and T. litoralis GDH by SDM. Both enzymes were stabilized by the newly introduced ion pair network (280,348). These studies confirmed the role of ion pair networks in the P. furiosus, P. kodakaraensis, and T. litoralis GDH thermostabilities.…”

Section: Ion Pairssupporting

confidence: 66%

“…5). The mutation Glu158Gln, which removed two ion pairs at the center of this network, significantly destabilized P. kodakaraensis GDH (280). One ion pair network involving six charged residues is present only in P. furiosus GDH.…”

Section: Ion Pairsmentioning

confidence: 99%

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

Vieille¹,

Zeikus

2001

Microbiol Mol Biol Rev

1,817

1,463

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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

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“…Improving thermostability by eliminating unfavorable electrostatic interactions has been reported previously for hyperthermostable CutA1 protein from Pyrococcus horikoshii (33). In addition, introduction of an ion pair network has been shown to improve the thermostability of glutamate dehydrogenases from Thermococcus litoralis (34) and Thermococcus kodakarensis KOD1 (35). Based on these results, electrostatic interaction optimization is an effective strategy to further stabilize hyperthermostable enzymes artificially.…”

Section: Discussionmentioning

confidence: 57%

Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus

Gao

Zeng

et al. 2017

Appl Environ Microbiol

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Pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus is the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily (subtilases). It contains four inserts (IS147, IS29, IS27, and IS8) of unknown function in the catalytic domain. We performed domain deletions and showed that three inserts are either essential (IS147 and IS27) or important (IS8) for efficient maturation of pyrolysin at high temperatures, whereas IS29 is dispensable. The large insert IS147 contains Ca3 and Ca4, two calcium-binding Dx [DN]xDG motifs that are conserved in many pyrolysin-like proteases. Mutagenesis revealed that the Ca3 site contributes to enzyme thermostability and the Ca4 site is necessary for pyrolysin to fold into a maturation-competent conformation. Mature insert-deletion variants were characterized and showed that IS29 and IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites: one within IS29 and the other in IS27. Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Interestingly, autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation. Additionally, a single mutation in IS29 increases resistance to salt-induced autocleavage and further increases enzyme thermostability. Our results suggest that these extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments. IMPORTANCE Pyrolysin-like proteases belong to the subtilase superfamily and are characterized by large inserts and long C-terminal extensions; however, the role of the inserts in enzyme function is unclear. Our results demonstrate that four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease.

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Ion Pairs Involved in Maintaining a Thermostable Structure of Glutamate Dehydrogenase from a Hyperthermophilic Archaeon

Cited by 50 publications

References 27 publications

Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca ²⁺ -Binding Site

Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca ²⁺ -Binding Site

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

Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus

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Ion Pairs Involved in Maintaining a Thermostable Structure of Glutamate Dehydrogenase from a Hyperthermophilic Archaeon

Cited by 50 publications

References 27 publications

Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca 2+ -Binding Site

Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca 2+ -Binding Site

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

Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus

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Product

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Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca ²⁺ -Binding Site

Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca ²⁺ -Binding Site