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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.

Show MeSH
Determination of optimum pH of activity 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 pH, as described in Materials and methods. Each data point is an average of three independent experiments having similar values.
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pone-0062619-g004: Determination of optimum pH of activity 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 pH, as described in Materials and methods. Each data point is an average of three independent experiments having similar values.

Mentions: Mutant enzymes show high proteolytic activity at neutral pH range (6.5–7.5) like wild-type (Fig. 4). All the three mutants have a broad range of pH optima (90–100% activity in the pH range 4.5–7.5) which can be of advantage for large scale production.


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

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

Determination of optimum pH of activity 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 pH, as described in Materials and methods. Each data point is an average of three independent experiments having similar values.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062619-g004: Determination of optimum pH of activity 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 pH, as described in Materials and methods. Each data point is an average of three independent experiments having similar values.
Mentions: Mutant enzymes show high proteolytic activity at neutral pH range (6.5–7.5) like wild-type (Fig. 4). All the three mutants have a broad range of pH optima (90–100% activity in the pH range 4.5–7.5) which can be of advantage for large scale production.

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