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Enhancement of proteolytic activity of a thermostable papain-like protease by structure-based rational design.

Dutta S, Dattagupta JK, Biswas S - PLoS ONE (2013)

Bottom Line: The double mutant does not achieve the catalytic efficiency of the template enzyme Ervatamin-A.By modeling the structure of the double mutant and probing the role of active site residues by docking a substrate, the mechanistic insights of higher activity of the mutant protease have been addressed.The in-silico study demonstrates that the residues beyond the catalytic cleft also influence the substrate binding and positioning of the substrate at the catalytic centre, thus controlling the catalytic efficiency of an enzyme.

View Article: PubMed Central - PubMed

Affiliation: Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.

ABSTRACT
Ervatamins (A, B and C) are papain-like cysteine proteases from the plant Ervatamia coronaria. Among Ervatamins, Ervatamin-C is a thermostable protease, but it shows lower catalytic efficiency. In contrast, Ervatamin-A which has a high amino acid sequence identity (∼90%) and structural homology (Cα rmsd 0.4 Å) with Ervatamin-C, has much higher catalytic efficiency (∼57 times). From the structural comparison of Ervatamin-A and -C, two residues Thr32 and Tyr67 in the catalytic cleft of Ervatamin-A have been identified whose contributions for higher activity of Ervatamin-A are established in our earlier studies. In this study, these two residues have been introduced in Ervatamin-C by site directed mutagenesis to enhance the catalytic efficiency of the thermostable protease. Two single mutants (S32T and A67Y) and one double mutant (S32T/A67Y) of Ervatamin-C have been generated and characterized. All the three mutants show ∼ 8 times higher catalytic efficiency (k cat/K m) than the wild-type. The thermostability of all the three mutant enzymes remained unchanged. The double mutant does not achieve the catalytic efficiency of the template enzyme Ervatamin-A. By modeling the structure of the double mutant and probing the role of active site residues by docking a substrate, the mechanistic insights of higher activity of the mutant protease have been addressed. The in-silico study demonstrates that the residues beyond the catalytic cleft also influence the substrate binding and positioning of the substrate at the catalytic centre, thus controlling the catalytic efficiency of an enzyme.

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Time course of activation of pro-enzymes to the mature and active form of the wild-type and the mutants of Erv-C.Aliquots of purified pro-enzymes (10–20 µg) were treated for activation for 0 to 50 minutes to convert into their respective mature forms and the percentage of residual enzyme activities were determined with respect to the maximum activity using an azocasein assay, as described in Materials and methods.
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pone-0062619-g003: Time course of activation of pro-enzymes to the mature and active form of the wild-type and the mutants of Erv-C.Aliquots of purified pro-enzymes (10–20 µg) were treated for activation for 0 to 50 minutes to convert into their respective mature forms and the percentage of residual enzyme activities were determined with respect to the maximum activity using an azocasein assay, as described in Materials and methods.

Mentions: The mutant pro-enzymes could be activated by using the same activator, buffer (20 mM cysteine in 50 mM Na-acetate buffer pH 5.00) and temperature (60°C) like wild-type Erv-C [17]. However, the time of activation of all the three mutant pro-enzymes significantly differs from the wild-type. S32T and S32T/A67Y could be activated by giving a short heat shock for 30 sec at 60°C while for A67Y, an incubation of 10 min at 60°C is required to get maximum activity. The activity of S32T sharply decreases with time while for other two mutants, activities decrease slowly. In contrast, wild-type required 30–40 min for reaching maximum activity and more than 90% activity is observed (Fig. 3) during the time range of 20–50 min.


Enhancement of proteolytic activity of a thermostable papain-like protease by structure-based rational design.

Dutta S, Dattagupta JK, Biswas S - PLoS ONE (2013)

Time course of activation of pro-enzymes to the mature and active form of the wild-type and the mutants of Erv-C.Aliquots of purified pro-enzymes (10–20 µg) were treated for activation for 0 to 50 minutes to convert into their respective mature forms and the percentage of residual enzyme activities were determined with respect to the maximum activity using an azocasein assay, as described in Materials and methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643963&req=5

pone-0062619-g003: Time course of activation of pro-enzymes to the mature and active form of the wild-type and the mutants of Erv-C.Aliquots of purified pro-enzymes (10–20 µg) were treated for activation for 0 to 50 minutes to convert into their respective mature forms and the percentage of residual enzyme activities were determined with respect to the maximum activity using an azocasein assay, as described in Materials and methods.
Mentions: The mutant pro-enzymes could be activated by using the same activator, buffer (20 mM cysteine in 50 mM Na-acetate buffer pH 5.00) and temperature (60°C) like wild-type Erv-C [17]. However, the time of activation of all the three mutant pro-enzymes significantly differs from the wild-type. S32T and S32T/A67Y could be activated by giving a short heat shock for 30 sec at 60°C while for A67Y, an incubation of 10 min at 60°C is required to get maximum activity. The activity of S32T sharply decreases with time while for other two mutants, activities decrease slowly. In contrast, wild-type required 30–40 min for reaching maximum activity and more than 90% activity is observed (Fig. 3) during the time range of 20–50 min.

Bottom Line: The double mutant does not achieve the catalytic efficiency of the template enzyme Ervatamin-A.By modeling the structure of the double mutant and probing the role of active site residues by docking a substrate, the mechanistic insights of higher activity of the mutant protease have been addressed.The in-silico study demonstrates that the residues beyond the catalytic cleft also influence the substrate binding and positioning of the substrate at the catalytic centre, thus controlling the catalytic efficiency of an enzyme.

View Article: PubMed Central - PubMed

Affiliation: Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.

ABSTRACT
Ervatamins (A, B and C) are papain-like cysteine proteases from the plant Ervatamia coronaria. Among Ervatamins, Ervatamin-C is a thermostable protease, but it shows lower catalytic efficiency. In contrast, Ervatamin-A which has a high amino acid sequence identity (∼90%) and structural homology (Cα rmsd 0.4 Å) with Ervatamin-C, has much higher catalytic efficiency (∼57 times). From the structural comparison of Ervatamin-A and -C, two residues Thr32 and Tyr67 in the catalytic cleft of Ervatamin-A have been identified whose contributions for higher activity of Ervatamin-A are established in our earlier studies. In this study, these two residues have been introduced in Ervatamin-C by site directed mutagenesis to enhance the catalytic efficiency of the thermostable protease. Two single mutants (S32T and A67Y) and one double mutant (S32T/A67Y) of Ervatamin-C have been generated and characterized. All the three mutants show ∼ 8 times higher catalytic efficiency (k cat/K m) than the wild-type. The thermostability of all the three mutant enzymes remained unchanged. The double mutant does not achieve the catalytic efficiency of the template enzyme Ervatamin-A. By modeling the structure of the double mutant and probing the role of active site residues by docking a substrate, the mechanistic insights of higher activity of the mutant protease have been addressed. The in-silico study demonstrates that the residues beyond the catalytic cleft also influence the substrate binding and positioning of the substrate at the catalytic centre, thus controlling the catalytic efficiency of an enzyme.

Show MeSH