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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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Analyses of thermal stability of the wild-type and the mutants of Erv-C.A. Determination of optimum temperature of activity (Topt) of the wild-type and the mutants of Erv-C. Purified pro-enzymes (10–20 µg) were converted to their respective mature forms and the percentage residual enzyme activities were determined with respect to the maximum activity using an azocasein assay at different temperatures as described in Materials and methods. B. Effect of temperature on activity of the wild type and the mutants of Erv-C. Each purified pro-enzyme (10–20 µg) was treated for 10 min at different temperatures followed by activation of the pro-enzymes to their respective mature forms. The percentage residual enzyme activities (at each temperature) were determined with respect to the maximum activity using an azocasein assay. Each data point is an average of three independent experiments having similar values for both the graphs.
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pone-0062619-g005: Analyses of thermal stability of the wild-type and the mutants of Erv-C.A. Determination of optimum temperature of activity (Topt) of the wild-type and the mutants of Erv-C. Purified pro-enzymes (10–20 µg) were converted to their respective mature forms and the percentage residual enzyme activities were determined with respect to the maximum activity using an azocasein assay at different temperatures as described in Materials and methods. B. Effect of temperature on activity of the wild type and the mutants of Erv-C. Each purified pro-enzyme (10–20 µg) was treated for 10 min at different temperatures followed by activation of the pro-enzymes to their respective mature forms. The percentage residual enzyme activities (at each temperature) were determined with respect to the maximum activity using an azocasein assay. Each data point is an average of three independent experiments having similar values for both the graphs.

Mentions: Operational stability of a biocatalyst is an important aspect for its use in different applications, particularly those which need large scale production. Enhancement of proteolytic activity of S32T, A67Y and S32T/A67Y mutants makes them promising enzymes for potential application in industry. To investigate whether the mutant enzymes with enhanced catalytic activity retain their thermal stability like wild-type, we measured and compared optimum temperature of activity (Topt) (Fig. 5A) and temperature of maximum activity (Tmax) (Fig. 5B) (Table 3). As shown in Figure 5A, the Topt analyses suggest that proteolytic activities of all the mutants are in the range of 65°–70°C which are similar to that of the wild-type. Temperatures of maximum proteolytic activity (Tmax) for S32T, A67Y and S32T/A67Y were 45°C, 40°C and 60°C respectively (Table 3). These findings indicate that the mutant enzymes in general retain thermal stability almost like their wild-type counterpart.


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

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

Analyses of thermal stability of the wild-type and the mutants of Erv-C.A. Determination of optimum temperature of activity (Topt) of the wild-type and the mutants of Erv-C. Purified pro-enzymes (10–20 µg) were converted to their respective mature forms and the percentage residual enzyme activities were determined with respect to the maximum activity using an azocasein assay at different temperatures as described in Materials and methods. B. Effect of temperature on activity of the wild type and the mutants of Erv-C. Each purified pro-enzyme (10–20 µg) was treated for 10 min at different temperatures followed by activation of the pro-enzymes to their respective mature forms. The percentage residual enzyme activities (at each temperature) were determined with respect to the maximum activity using an azocasein assay. Each data point is an average of three independent experiments having similar values for both the graphs.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643963&req=5

pone-0062619-g005: Analyses of thermal stability of the wild-type and the mutants of Erv-C.A. Determination of optimum temperature of activity (Topt) of the wild-type and the mutants of Erv-C. Purified pro-enzymes (10–20 µg) were converted to their respective mature forms and the percentage residual enzyme activities were determined with respect to the maximum activity using an azocasein assay at different temperatures as described in Materials and methods. B. Effect of temperature on activity of the wild type and the mutants of Erv-C. Each purified pro-enzyme (10–20 µg) was treated for 10 min at different temperatures followed by activation of the pro-enzymes to their respective mature forms. The percentage residual enzyme activities (at each temperature) were determined with respect to the maximum activity using an azocasein assay. Each data point is an average of three independent experiments having similar values for both the graphs.
Mentions: Operational stability of a biocatalyst is an important aspect for its use in different applications, particularly those which need large scale production. Enhancement of proteolytic activity of S32T, A67Y and S32T/A67Y mutants makes them promising enzymes for potential application in industry. To investigate whether the mutant enzymes with enhanced catalytic activity retain their thermal stability like wild-type, we measured and compared optimum temperature of activity (Topt) (Fig. 5A) and temperature of maximum activity (Tmax) (Fig. 5B) (Table 3). As shown in Figure 5A, the Topt analyses suggest that proteolytic activities of all the mutants are in the range of 65°–70°C which are similar to that of the wild-type. Temperatures of maximum proteolytic activity (Tmax) for S32T, A67Y and S32T/A67Y were 45°C, 40°C and 60°C respectively (Table 3). These findings indicate that the mutant enzymes in general retain thermal stability almost like their wild-type counterpart.

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