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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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Comparison of Erv-A and -C.A. Amino acid sequence alignment of Erv-A and –C. Similar residues and mismatched residues are shaded yellow and sky blue. The mismatched residues in the catalytic clefts which are targets for mutation are indicated by red stars. B. Structural superposition of Erv-A and –C. The structures of Erv-A and –C are represented by Cα traces in orange and green colours respectively. The mismatched residues in the catalytic clefts are represented by ball and stick models. The catalytic dyad residues Cys25 and His 157 of Erv-A are presented as spheres.
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pone-0062619-g001: Comparison of Erv-A and -C.A. Amino acid sequence alignment of Erv-A and –C. Similar residues and mismatched residues are shaded yellow and sky blue. The mismatched residues in the catalytic clefts which are targets for mutation are indicated by red stars. B. Structural superposition of Erv-A and –C. The structures of Erv-A and –C are represented by Cα traces in orange and green colours respectively. The mismatched residues in the catalytic clefts are represented by ball and stick models. The catalytic dyad residues Cys25 and His 157 of Erv-A are presented as spheres.

Mentions: For rational design of new mutants of Erv-C with an improved catalytic efficiency, it is important to explore the major factors affecting the catalytic properties of Erv-C and if possible to understand the correlation between the structure and catalytic efficiency of the protease. In the present study, we have used the structure of Ervatamin-A (Erv-A), another papain-like protease from the same source having ∼90% amino acid sequence identity (Fig. 1A) but with much higher catalytic efficiency compared to Erv-C [16], as a template to identify the factors responsible for higher catalytic efficiency. Two homologous 3D structures of Erv-A and Erv-C (Fig. 1B) have been compared to identify the mutations in the catalytic cleft which need to be incorporated in Erv-C to enhance its proteolytic activity. The mutants were generated and characterized experimentally which showed higher catalytic efficiency compared to wild-type. We also aimed to explore the structure-activity correlation by modelling the structure of enzyme-substrate complexes of Erv-A, Erv-C and the double mutant of Erv-C. The combined structural and experimental results demonstrate a reasonable correlation between the structure and the catalytic efficiency of the enzyme.


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

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

Comparison of Erv-A and -C.A. Amino acid sequence alignment of Erv-A and –C. Similar residues and mismatched residues are shaded yellow and sky blue. The mismatched residues in the catalytic clefts which are targets for mutation are indicated by red stars. B. Structural superposition of Erv-A and –C. The structures of Erv-A and –C are represented by Cα traces in orange and green colours respectively. The mismatched residues in the catalytic clefts are represented by ball and stick models. The catalytic dyad residues Cys25 and His 157 of Erv-A are presented as spheres.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643963&req=5

pone-0062619-g001: Comparison of Erv-A and -C.A. Amino acid sequence alignment of Erv-A and –C. Similar residues and mismatched residues are shaded yellow and sky blue. The mismatched residues in the catalytic clefts which are targets for mutation are indicated by red stars. B. Structural superposition of Erv-A and –C. The structures of Erv-A and –C are represented by Cα traces in orange and green colours respectively. The mismatched residues in the catalytic clefts are represented by ball and stick models. The catalytic dyad residues Cys25 and His 157 of Erv-A are presented as spheres.
Mentions: For rational design of new mutants of Erv-C with an improved catalytic efficiency, it is important to explore the major factors affecting the catalytic properties of Erv-C and if possible to understand the correlation between the structure and catalytic efficiency of the protease. In the present study, we have used the structure of Ervatamin-A (Erv-A), another papain-like protease from the same source having ∼90% amino acid sequence identity (Fig. 1A) but with much higher catalytic efficiency compared to Erv-C [16], as a template to identify the factors responsible for higher catalytic efficiency. Two homologous 3D structures of Erv-A and Erv-C (Fig. 1B) have been compared to identify the mutations in the catalytic cleft which need to be incorporated in Erv-C to enhance its proteolytic activity. The mutants were generated and characterized experimentally which showed higher catalytic efficiency compared to wild-type. We also aimed to explore the structure-activity correlation by modelling the structure of enzyme-substrate complexes of Erv-A, Erv-C and the double mutant of Erv-C. The combined structural and experimental results demonstrate a reasonable correlation between the structure and the catalytic efficiency of the enzyme.

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
Related in: MedlinePlus