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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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SDS–PAGE analysis and gelatin gel zymography.A. Purified and refolded pro-enzymes of mutants and wild-type were analyzed in 15% SDS-PAGE; M denotes Molecular mass markers. B. Gelatin gel assay of the activated mutants (∼15 µg) and wild-type enzymes.
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pone-0062619-g002: SDS–PAGE analysis and gelatin gel zymography.A. Purified and refolded pro-enzymes of mutants and wild-type were analyzed in 15% SDS-PAGE; M denotes Molecular mass markers. B. Gelatin gel assay of the activated mutants (∼15 µg) and wild-type enzymes.

Mentions: Purification and refolding of the recombinantly expressed mutant enzymes were confirmed by SDS-PAGE (Fig. 2A). The conditions, efficiency of refolding and the yield were similar for all the mutant enzymes and the wild-type [17]. The proteolytic activities of the refolded proteins were verified by zymography (Fig. 2B).


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

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

SDS–PAGE analysis and gelatin gel zymography.A. Purified and refolded pro-enzymes of mutants and wild-type were analyzed in 15% SDS-PAGE; M denotes Molecular mass markers. B. Gelatin gel assay of the activated mutants (∼15 µg) and wild-type enzymes.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062619-g002: SDS–PAGE analysis and gelatin gel zymography.A. Purified and refolded pro-enzymes of mutants and wild-type were analyzed in 15% SDS-PAGE; M denotes Molecular mass markers. B. Gelatin gel assay of the activated mutants (∼15 µg) and wild-type enzymes.
Mentions: Purification and refolding of the recombinantly expressed mutant enzymes were confirmed by SDS-PAGE (Fig. 2A). The conditions, efficiency of refolding and the yield were similar for all the mutant enzymes and the wild-type [17]. The proteolytic activities of the refolded proteins were verified by zymography (Fig. 2B).

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