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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 three dimensional structures of Erv-A, Erv-C and double mutant of Erv-C (S32T/A67Y).A, B, and C. Surface presentation of Erv-A, Erv-C and double mutant of Erv-C. The residues in positions 32, 67 and catalytic cysteins are presented in ball and stick model. D. Overlay of the three dimensional structures of the three enzymes; sky-blue, magenta and green colored cartoons are for Erv-A, Erv-C and double mutant of Erv-C. The important residues are labeled and represented in stick model. Distances of the catalytic dyad (C25SG and H157ND1) of the three enzymes are marked. E. Ramachandran plot highlighting G66 residues in three enzymes (1:Erv-A, 2: Erv-C and 3:Erv-C double mutant). The red colored points are for the double mutant of Erv-C.
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pone-0062619-g006: Comparison of three dimensional structures of Erv-A, Erv-C and double mutant of Erv-C (S32T/A67Y).A, B, and C. Surface presentation of Erv-A, Erv-C and double mutant of Erv-C. The residues in positions 32, 67 and catalytic cysteins are presented in ball and stick model. D. Overlay of the three dimensional structures of the three enzymes; sky-blue, magenta and green colored cartoons are for Erv-A, Erv-C and double mutant of Erv-C. The important residues are labeled and represented in stick model. Distances of the catalytic dyad (C25SG and H157ND1) of the three enzymes are marked. E. Ramachandran plot highlighting G66 residues in three enzymes (1:Erv-A, 2: Erv-C and 3:Erv-C double mutant). The red colored points are for the double mutant of Erv-C.

Mentions: It is established that both native Erv-A and Erv-C, purified from the latex of the plant, prefer a branched hydrophobic side chain (Val or Leu) at the P2 position of a substrate [16]. Of the five residues (Tyr67, Phe68, Ala131, Leu155 and Leu201) forming the S2 subsite cleft of Erv-A one substitution (Tyr67 is replaced by Ala67) is observed in Erv-C [16]. The side chain of Tyr67 in Erv-A points towards the interface of S2 and S3 subsites, providing tight packing and a favourable hydrophobic environment for P2 position of a substrate at the S2 subsite cleft [16] (Fig. 6A). Due to Tyr67→Ala67 replacement, the S2 cavity of Erv-C has a wider opening than that of Erv-A and as a result lacks the proper environment to bind and fix a hydrophobic side chain (P2) of a substrate [16] (Fig. 6B).


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 three dimensional structures of Erv-A, Erv-C and double mutant of Erv-C (S32T/A67Y).A, B, and C. Surface presentation of Erv-A, Erv-C and double mutant of Erv-C. The residues in positions 32, 67 and catalytic cysteins are presented in ball and stick model. D. Overlay of the three dimensional structures of the three enzymes; sky-blue, magenta and green colored cartoons are for Erv-A, Erv-C and double mutant of Erv-C. The important residues are labeled and represented in stick model. Distances of the catalytic dyad (C25SG and H157ND1) of the three enzymes are marked. E. Ramachandran plot highlighting G66 residues in three enzymes (1:Erv-A, 2: Erv-C and 3:Erv-C double mutant). The red colored points are for the double mutant of Erv-C.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643963&req=5

pone-0062619-g006: Comparison of three dimensional structures of Erv-A, Erv-C and double mutant of Erv-C (S32T/A67Y).A, B, and C. Surface presentation of Erv-A, Erv-C and double mutant of Erv-C. The residues in positions 32, 67 and catalytic cysteins are presented in ball and stick model. D. Overlay of the three dimensional structures of the three enzymes; sky-blue, magenta and green colored cartoons are for Erv-A, Erv-C and double mutant of Erv-C. The important residues are labeled and represented in stick model. Distances of the catalytic dyad (C25SG and H157ND1) of the three enzymes are marked. E. Ramachandran plot highlighting G66 residues in three enzymes (1:Erv-A, 2: Erv-C and 3:Erv-C double mutant). The red colored points are for the double mutant of Erv-C.
Mentions: It is established that both native Erv-A and Erv-C, purified from the latex of the plant, prefer a branched hydrophobic side chain (Val or Leu) at the P2 position of a substrate [16]. Of the five residues (Tyr67, Phe68, Ala131, Leu155 and Leu201) forming the S2 subsite cleft of Erv-A one substitution (Tyr67 is replaced by Ala67) is observed in Erv-C [16]. The side chain of Tyr67 in Erv-A points towards the interface of S2 and S3 subsites, providing tight packing and a favourable hydrophobic environment for P2 position of a substrate at the S2 subsite cleft [16] (Fig. 6A). Due to Tyr67→Ala67 replacement, the S2 cavity of Erv-C has a wider opening than that of Erv-A and as a result lacks the proper environment to bind and fix a hydrophobic side chain (P2) of a substrate [16] (Fig. 6B).

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