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Half-Barrels Derived from a (β/α)8 Barrel β-Glycosidase Undergo an Activation Process.

Beton D, Marana SR - PLoS ONE (2015)

Bottom Line: The rate constants of the activation process were calculated to be 0.029 and 0.032 h-1 for Sfβgly-N and Sfβgly-C, respectively.Importantly, this activation was also coincident with an increase in the sizes of Sfβgly-N and Sfβgly-C particles.These novel observations suggest that the change in catalytic activity associated with the transition from a half to whole (β/α)8 barrel might also have driven such an evolutionary process.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

ABSTRACT
The evolution of (β/α)8 barrel proteins is currently thought to have involved the fusion of two (β/α)4 half-barrels, thereby conferring stability on the protein structure. After the formation of a whole (β/α)8 barrel, this structure could evolve and diverge to form fully active enzymes. Interestingly, we show here that isolated (β/α)4 half-barrels derived from the N- and C-terminal domains of the β-glucosidase Sfβgly (Sfβgly-N: residues 1 to 265; Sfβgly-C: residues 266 to 509) undergo an activation process, which renders them catalytically active. The rate constants of the activation process were calculated to be 0.029 and 0.032 h-1 for Sfβgly-N and Sfβgly-C, respectively. Moreover, the Sfβgly-N and Sfβgly-C activation processes were simultaneous with modifications in their initial structure, which reduced the exposure of their tryptophan residues. Importantly, this activation was also coincident with an increase in the sizes of Sfβgly-N and Sfβgly-C particles. These novel observations suggest that the change in catalytic activity associated with the transition from a half to whole (β/α)8 barrel might also have driven such an evolutionary process.

No MeSH data available.


Related in: MedlinePlus

Enzyme activity assay of Sfβgly-N and Sfβgly-C showing an activation process.Purified samples of Sfβgly-N (A) and Sfβgly-C (B) in 100 mM citrate-phosphate buffer, pH 6.0, were incubated with substrate 1 mM methylumbelliferyl β-glucoside prepared in 100 mM citrate-phosphate buffer, pH 6.0, for 16, 22, 40 and 48 h. The fluorescence of the product methylumbelliferone (excitation, 360 nm; emission, 450 nm) was determined after the addition of 100 mM glycine-NaOH, pH 10.5. Inserts: rates were analyzed according the hysteresis model [10]. The reaction rate at each time (vt) was calculated based on the slope at that specific time. The slope at 48 h was taken as the final rate (v2).
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pone.0139673.g002: Enzyme activity assay of Sfβgly-N and Sfβgly-C showing an activation process.Purified samples of Sfβgly-N (A) and Sfβgly-C (B) in 100 mM citrate-phosphate buffer, pH 6.0, were incubated with substrate 1 mM methylumbelliferyl β-glucoside prepared in 100 mM citrate-phosphate buffer, pH 6.0, for 16, 22, 40 and 48 h. The fluorescence of the product methylumbelliferone (excitation, 360 nm; emission, 450 nm) was determined after the addition of 100 mM glycine-NaOH, pH 10.5. Inserts: rates were analyzed according the hysteresis model [10]. The reaction rate at each time (vt) was calculated based on the slope at that specific time. The slope at 48 h was taken as the final rate (v2).

Mentions: Sfβgly-N and Sfβgly-C were subjected to enzymatic activity assays using methylumbelliferyl β-glucoside as a substrate. Remarkably, both half-barrels exhibited hydrolytic activity toward that substrate, which increased with time and yielded exponential curves (Fig 2). These findings suggest that the concentration of catalytically active Sfβgly-N and Sfβgly-C increased with assay time, indicating that these half-barrels underwent an activation process. Native Sfβgly did not exhibit such behavior in similar activity assays actually generating linear curves as expected. Moreover, control assays prepared with substrate alone, in which the half-barrel samples were replaced by buffer containing 300 mM imidazole,did not show β-glucosidase activity. Finally, mock “Sfβgly-N and Sfβgly-C samples” produced from extracts of ArticExpress (DE3) bacteria transformed with an “empty” pAE vector were used in the β-glucosidase assays, and these assays showed no product formation. Accordingly, mock “Sfβgly-N and Sfβgly-C samples” produced from ArticExpress (DE3) bacteria expressing an inactive mutant Sfβgly showed no product formation.


Half-Barrels Derived from a (β/α)8 Barrel β-Glycosidase Undergo an Activation Process.

Beton D, Marana SR - PLoS ONE (2015)

Enzyme activity assay of Sfβgly-N and Sfβgly-C showing an activation process.Purified samples of Sfβgly-N (A) and Sfβgly-C (B) in 100 mM citrate-phosphate buffer, pH 6.0, were incubated with substrate 1 mM methylumbelliferyl β-glucoside prepared in 100 mM citrate-phosphate buffer, pH 6.0, for 16, 22, 40 and 48 h. The fluorescence of the product methylumbelliferone (excitation, 360 nm; emission, 450 nm) was determined after the addition of 100 mM glycine-NaOH, pH 10.5. Inserts: rates were analyzed according the hysteresis model [10]. The reaction rate at each time (vt) was calculated based on the slope at that specific time. The slope at 48 h was taken as the final rate (v2).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591997&req=5

pone.0139673.g002: Enzyme activity assay of Sfβgly-N and Sfβgly-C showing an activation process.Purified samples of Sfβgly-N (A) and Sfβgly-C (B) in 100 mM citrate-phosphate buffer, pH 6.0, were incubated with substrate 1 mM methylumbelliferyl β-glucoside prepared in 100 mM citrate-phosphate buffer, pH 6.0, for 16, 22, 40 and 48 h. The fluorescence of the product methylumbelliferone (excitation, 360 nm; emission, 450 nm) was determined after the addition of 100 mM glycine-NaOH, pH 10.5. Inserts: rates were analyzed according the hysteresis model [10]. The reaction rate at each time (vt) was calculated based on the slope at that specific time. The slope at 48 h was taken as the final rate (v2).
Mentions: Sfβgly-N and Sfβgly-C were subjected to enzymatic activity assays using methylumbelliferyl β-glucoside as a substrate. Remarkably, both half-barrels exhibited hydrolytic activity toward that substrate, which increased with time and yielded exponential curves (Fig 2). These findings suggest that the concentration of catalytically active Sfβgly-N and Sfβgly-C increased with assay time, indicating that these half-barrels underwent an activation process. Native Sfβgly did not exhibit such behavior in similar activity assays actually generating linear curves as expected. Moreover, control assays prepared with substrate alone, in which the half-barrel samples were replaced by buffer containing 300 mM imidazole,did not show β-glucosidase activity. Finally, mock “Sfβgly-N and Sfβgly-C samples” produced from extracts of ArticExpress (DE3) bacteria transformed with an “empty” pAE vector were used in the β-glucosidase assays, and these assays showed no product formation. Accordingly, mock “Sfβgly-N and Sfβgly-C samples” produced from ArticExpress (DE3) bacteria expressing an inactive mutant Sfβgly showed no product formation.

Bottom Line: The rate constants of the activation process were calculated to be 0.029 and 0.032 h-1 for Sfβgly-N and Sfβgly-C, respectively.Importantly, this activation was also coincident with an increase in the sizes of Sfβgly-N and Sfβgly-C particles.These novel observations suggest that the change in catalytic activity associated with the transition from a half to whole (β/α)8 barrel might also have driven such an evolutionary process.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

ABSTRACT
The evolution of (β/α)8 barrel proteins is currently thought to have involved the fusion of two (β/α)4 half-barrels, thereby conferring stability on the protein structure. After the formation of a whole (β/α)8 barrel, this structure could evolve and diverge to form fully active enzymes. Interestingly, we show here that isolated (β/α)4 half-barrels derived from the N- and C-terminal domains of the β-glucosidase Sfβgly (Sfβgly-N: residues 1 to 265; Sfβgly-C: residues 266 to 509) undergo an activation process, which renders them catalytically active. The rate constants of the activation process were calculated to be 0.029 and 0.032 h-1 for Sfβgly-N and Sfβgly-C, respectively. Moreover, the Sfβgly-N and Sfβgly-C activation processes were simultaneous with modifications in their initial structure, which reduced the exposure of their tryptophan residues. Importantly, this activation was also coincident with an increase in the sizes of Sfβgly-N and Sfβgly-C particles. These novel observations suggest that the change in catalytic activity associated with the transition from a half to whole (β/α)8 barrel might also have driven such an evolutionary process.

No MeSH data available.


Related in: MedlinePlus