Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Cacciapuoti, Giovanna; Fuccio, Francesca; Petraccone, Luigi; Vecchio, Pompea Del; Porcelli, Marina

doi:10.1016/j.bbapap.2012.06.014

Cited by 19 publications

(11 citation statements)

References 38 publications

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“…Within thermostable enzymes these structural elements are significant, since they have been shown to increase stability within thermophilic proteins and play a role in preventing alteration of quaternary structure. Studies by Cacciapuoti et al [12, 33] and separately by Boutz et al [34] provide evidence for these claims.…”

Section: Thermophilic Proteinsmentioning

confidence: 98%

“…One example of the use of disulfide bridging in thermostability is 5′-deoxy-5′methylthioadenosine phosphorylase II which was used to study the CXC motif and intrasubunit disulfide bonds within thermophilic proteins [12, 33]. Using circular dichroism spectroscopy, under reducing conditions, the hexameric protein was seen to disassociate into its monomeric state in a reversible fashion.…”

Section: Thermophilic Proteinsmentioning

confidence: 99%

“…The native protein was observed to be almost completely denatured at 108°C, whereas the single mutant (C262S) was shifted to 102°C and 99°C for the CXC double mutant (C259S/C261S). These results elucidated that mutations within these residues decreased thermal stability inferring that disulfide bridging is a structural adaptation [12]. It is noteworthy that the CXC motif forms a strained 11-member disulfide ring, which has been implicated as a useful redox reagent [35].…”

Section: Thermophilic Proteinsmentioning

confidence: 99%

“…This makes studying protein adaptations in all extremophiles, especially Archaea, difficult because one is not simply looking for a single trend or feature. In fact, variability in adaptations has been noted multiple times throughout studies of archaeal and extremophile proteins [4, 12–14]. There have been many reasons proposed for these differences.…”

Section: Introductionmentioning

confidence: 99%

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Protein Adaptations in Archaeal Extremophiles

Reed

Lewis

Trejo

et al. 2013

Archaea

259

174

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Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures. Psychrophilic proteins have a reduced hydrophobic core and a less charged protein surface to maintain flexibility and activity under cold temperatures. Halophilic proteins are characterized by increased negative surface charge due to increased acidic amino acid content and peptide insertions, which compensates for the extreme ionic conditions. While acidophiles, alkaliphiles, and piezophiles are their own class of Archaea, their protein adaptations toward pH and pressure are less discernible. By understanding the protein adaptations used by archaeal extremophiles, we hope to be able to engineer and utilize proteins for industrial, environmental, and biotechnological applications where function in extreme conditions is required for activity.

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Section: Thermophilic Proteinsmentioning

confidence: 98%

Section: Thermophilic Proteinsmentioning

confidence: 99%

Section: Thermophilic Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Protein Adaptations in Archaeal Extremophiles

Reed

Lewis

Trejo

et al. 2013

Archaea

259

174

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“…2 A ), the contribution of hydrophobic packing to thermal stability in KOD Pol, Pfu Pol, and BR3 Pol is unclear. Disulfide bonds could be a key adaptive feature in extremely thermostable polymerases ( 15 , 45 ). BR3 Pol retained its activity in temperatures up to ∼55°C ( Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep‐sea brines of the Red Sea

Takahashi

Joudeh

et al. 2018

FASEB j.

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The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein’s surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing. Salt bridge interactions via increased negative and positive charges contribute to structural stability. Salt tolerance, conversely, is mediated by a dynamic structure that becomes more fixed and functional with increasing salt concentration. We propose that repulsive forces among excess negative charges, in addition to a high percentage of negatively charged random coils, mediate this structural dynamism. This knowledge enabled us to engineer a halophilic version of Thermococcus kodakarensis DNA polymerase.—Takahashi, M., Takahashi, E., Joudeh, L. I., Marini, M., Das, G., Elshenawy, M. M., Akal, A., Sakashita, K., Alam, I., Tehseen, M., Sobhy, M. A., Stingl, U., Merzaban, J. S., Di Fabrizio, E., Hamdan, S. M. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

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Efficient Fludarabine‐Activating PNP From Archaea as a Guidance for Redesign the Active Site of E. Coli PNP

Cacciapuoti

Bagarolo

Martino

et al. 2015

J of Cellular Biochemistry

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The combination of the gene of purine nucleoside phosphorylase (PNP) from Escherichia coli and fludarabine represents one of the most promising systems in the gene therapy of solid tumors. The use of fludarabine in gene therapy is limited by the lack of an enzyme that is able to efficiently activate this prodrug which, consequently, has to be administered in high doses that cause serious side effects. In an attempt to identify enzymes with a better catalytic efficiency than E. coli PNP towards fludarabine to be used as a guidance on how to improve the activity of the bacterial enzyme, we have selected 5'-deoxy-5'-methylthioadenosine phosphorylase (SsMTAP) and 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII), two PNPs isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. Substrate specificity and catalytic efficiency of SsMTAP and SsMTAPII for fludarabine were analyzed by kinetic studies and compared with E. coli PNP. SsMTAP and SsMTAPII share with E. coli PNP a comparable low affinity for the arabinonucleoside but are better catalysts of fludarabine cleavage with k(cat)/K(m) values that are 12.8-fold and 6-fold higher, respectively, than those reported for the bacterial enzyme. A computational analysis of the interactions of fludarabine in the active sites of E. coli PNP, SsMTAP, and SsMTAPII allowed to identify the crucial residues involved in the binding with this substrate, and provided structural information to improve the catalytic efficiency of E. coli PNP by enzyme redesign.

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Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Cited by 19 publications

References 38 publications

Protein Adaptations in Archaeal Extremophiles

Protein Adaptations in Archaeal Extremophiles

Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep‐sea brines of the Red Sea

Efficient Fludarabine‐Activating PNP From Archaea as a Guidance for Redesign the Active Site of E. Coli PNP

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