Biochemical characterization and homology modeling of a purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus: Insights into mechanisms of protein stabilization

Porcelli, Marina; Peluso, Iolanda; Facchiano, Angelo; Cacciapuoti, Giovanna

doi:10.1016/j.abb.2008.12.005

Cited by 12 publications

(24 citation statements)

References 49 publications

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“…Incubation of the enzyme S-adenosylhomocysteine hydrolase from the hyperthermophile P. furiosus at 80 • C in 0.8 M dithiothreitol for 1 h resulted in a 63% loss of activity, which indicates that the Cys residues within this enzyme may be involved in disulfide linkages (87). Similar results were found in a study of purine-specific ribonucleoside hydrolase from the thermoacidophile Sulfolobus solfataricus, further proof that disulfide bonds may be an important structural mechanism for enzyme stability at high temperatures (88,89). Interestingly, denaturation of the disulfide bonds of the psychrophilic α-amylase from Pseudoaltermonas haloplanktis by β-mercaptoethanol reduced activity more than stability (84).…”

Section: Covalent Interactionssupporting

confidence: 74%

Nature Versus Nurture: Developing Enzymes That Function Under Extreme Conditions

Liszka¹,

Clark

Schneider

et al. 2012

Annu. Rev. Chem. Biomol. Eng.

176

116

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Many industrial processes used to produce chemicals and pharmaceuticals would benefit from enzymes that function under extreme conditions. Enzymes from extremophilic microorganisms have evolved to function in a variety of extreme environments, and bioprospecting for these microorganisms has led to the discovery of new enzymes with high tolerance to nonnatural conditions. However, bioprospecting is inherently limited by the diversity of enzymes evolved by nature. Protein engineering has also been successful in generating extremophilic enzymes by both rational mutagenesis and directed evolution, but screening for activity under extreme conditions can be difficult. This review examines the emerging synergy between bioprospecting and protein engineering in developing extremophilic enzymes. Specific topics include unnatural industrial conditions relevant to biocatalysis, biophysical properties of extremophilic enzymes, and industrially relevant extremophilic enzymes found either in nature or through protein engineering. 77 Annu. Rev. Chem. Biomol. Eng. 2012.3:77-102. Downloaded from www.annualreviews.org by University of Toronto on 11/15/12. For personal use only. Click here for quick links to Annual Reviews content online, including: • Other articles in this volume • Top cited articles • Top downloaded articles • Our comprehensive search Further ANNUAL REVIEWS T opt : optimum activity temperature 78 Liszka et al. Annu. Rev. Chem. Biomol. Eng. 2012.3:77-102. Downloaded from www.annualreviews.org by University of Toronto on 11/15/12. For personal use only. www.annualreviews.org • Enzymes Under Extreme Conditions 79 Annu. Rev. Chem. Biomol. Eng. 2012.3:77-102. Downloaded from www.annualreviews.org by University of Toronto on 11/15/12. For personal use only.greater potential for success. The answer to this question is not obvious and may depend strongly on the conditions required, the desired enzymatic reaction, and the type of enzyme used. EXTREME CONDITIONS RELEVANT TO INDUSTRIAL PROCESSES Conditions Found in NatureMicroorganisms exist in very different environments, and their enzymes and proteins have adapted to extreme temperatures, pressures, alkalinity/acidity, and/or osmolarity. Many of these extreme conditions mimic those found in industrial processes that currently employ enzymes or stand to benefit from them. Nature, therefore, is an abundant source of enzymes and proteins tolerant to extreme conditions. Extreme temperatures. Performing enzyme reactions at elevated temperatures has several potential advantages including higher substrate solubility, faster reaction rates, reduced risk of system contamination, lower solution viscosity, and increased solvent miscibility. However, there are many examples of enzymes used in processes that operate at lower temperatures as well, such as cold-active hydrolytic enzymes used in laundry detergents or for cleaning animal hides, proteases used for cleaning contact lenses, and pectinases for clarifying and extracting fruit juices (9). Mesophilic enzymes are less effective at...

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Section: Covalent Interactionssupporting

confidence: 74%

Nature Versus Nurture: Developing Enzymes That Function Under Extreme Conditions

Liszka¹,

Clark

Schneider

et al. 2012

Annu. Rev. Chem. Biomol. Eng.

176

116

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“…Furthermore, critical analysis of the active site noncovalent contacts highlights the importance of enzyme–DNA π–π stacking and T-shaped interactions, as well as the crucial role of water, in stabilizing the departing nucleobase. Overall, our study contributes to our understanding of the activity of enzymes with flexible actives sites and broad, yet discriminatory, substrate specificity, such as other DNA glycosylases ,,,− and nucleoside hydrolases. − …”

Section: Introductionmentioning

confidence: 84%

QM/MM Study of the Reaction Catalyzed by Alkyladenine DNA Glycosylase: Examination of the Substrate Specificity of a DNA Repair Enzyme

Lenz

Wetmore

2017

J. Phys. Chem. B

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Human alkyladenine DNA glycosylase (AAG) functions as part of the base excision repair pathway to excise structurally diverse oxidized and alkylated DNA purines. Specifically, AAG uses a water molecule activated by a general base and a nonspecific active site lined with aromatic residues to cleave the N-glycosidic bond. Despite broad substrate specificity, AAG does not target the natural purines (adenine (A) and guanine (G)). Using the ONIOM(QM:MM) methodology, we provide fundamental atomic level details of AAG bound to DNA-containing a neutral substrate (hypoxanthine (Hx)), a nonsubstrate (G), or a cationic substrate (7-methylguanine (7MeG)) and probe changes in the reaction pathway that occur when AAG targets different nucleotides. We reveal that subtle differences in protein-DNA contacts upon binding different substrates within the flexible AAG active site can significantly affect the deglycosylation reaction. Notably, we predict that AAG excises Hx in a concerted mechanism that is facilitated through correct alignment of the (E125) general base due to hydrogen bonding with a neighboring aromatic amino acid (Y127). Hx departure is further stabilized by π-π interactions with aromatic amino acids and hydrogen bonds with active site water. Despite possessing a similar structure to Hx, G is not excised since the additional exocyclic amino group leads to misalignment of the general base due to disruption of the key E125-Y127 hydrogen bond, the catalytically unfavorable placement of water within the active site, and weakened π-contacts between aromatic amino acids and the nucleobase. In contrast, cationic 7MeG does not occupy the same position within the AAG active site as G due to steric clashes with the additional N7 methyl group, which results in the correct alignment of the general base and permits nucleobase excision as observed for neutral Hx. Overall, our structural data rationalizes the observed substrate specificity of AAG and contributes to our fundamental understanding of enzymes with flexible active sites and broad substrate specificities.

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“…An NH-like gene ( iunH ) is present in the Mycobacterium tuberculosis genome, but mycobacteria engineered to prevent iunH gene expression are viable and infectious, thus indicating that the NH enzyme is not involved in key steps of the pathogen’s life cycle [ 44 , 45 ]. The archaeon Sulfolobus solfataricus also bears two NH-like genes, encoding for one purine-specific and one pyrimidine-specific enzyme endowed with very high thermal stability [ 46 , 47 ]. A growing number of studies are demonstrating how the NH structural scaffold can be used for other purposes than riboside hydrolysis.…”

Section: Reviewmentioning

confidence: 99%

“…The IAG-NH and CU-NH from the archaeon S. solfataricus also display peculiarities that make them exceptions to the principles inferred from the other NHs. The S. solfataricus IAG-NH is unusual in its kinetic parameters, in particular, K M values that are 4- to 60-fold larger compared to the trypanosomal IAG-NHs, making it more similar to IU-NHs [ 46 ]. The crystal structure of the enzyme indeed showed a group-I fold, and surprisingly, neither Trp nor His residue preceding the last Ca 2+ -coordinating Asp were poised for leaving-group activation [ 73 ].…”

Section: Reviewmentioning

confidence: 99%

“…Analysis of this surface using PISA shows that the interaction is generally computed as weaker, albeit with a wide range of energies, and more likely to dissociate in solution compared to the major interface ( Table S3 ). Thus, it can be envisioned that group I NHs in the cell, under dilute conditions, may exist as dimers as seen for the S. solfataricus IAGNH [ 46 ]. Nevertheless, it is noteworthy that all group I NHs from bacteria and protozoa and group III NHs from protozoa and metazoa so far characterized crystals as structurally homologous homotetramers, taking advantage of the same minor surface to form the dimer of dimers and with similar overall geometries.…”

Section: Reviewmentioning

confidence: 99%

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Structure, Oligomerization and Activity Modulation in N-Ribohydrolases

Degano

2022

IJMS

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Enzymes catalyzing the hydrolysis of the N-glycosidic bond in nucleosides and other ribosides (N-ribohydrolases, NHs) with diverse substrate specificities are found in all kingdoms of life. While the overall NH fold is highly conserved, limited substitutions and insertions can account for differences in substrate selection, catalytic efficiency, and distinct structural features. The NH structural module is also employed in monomeric proteins devoid of enzymatic activity with different physiological roles. The homo-oligomeric quaternary structure of active NHs parallels the different catalytic strategies used by each isozyme, while providing a buttressing effect to maintain the active site geometry and allow the conformational changes required for catalysis. The unique features of the NH catalytic strategy and structure make these proteins attractive targets for diverse therapeutic goals in different diseases.

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Biochemical characterization and homology modeling of a purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus: Insights into mechanisms of protein stabilization

Cited by 12 publications

References 49 publications

Nature Versus Nurture: Developing Enzymes That Function Under Extreme Conditions

Nature Versus Nurture: Developing Enzymes That Function Under Extreme Conditions

QM/MM Study of the Reaction Catalyzed by Alkyladenine DNA Glycosylase: Examination of the Substrate Specificity of a DNA Repair Enzyme

Structure, Oligomerization and Activity Modulation in N-Ribohydrolases

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