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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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Substrate docked at the active site cleft.Overlay of 100 poses (generated after minimization of initial 100 conformations of MD trajectory) of the substrate (N-benzoyl-Phe-Val-Arg-↓-pNA) docked at the active sites of A. Erv-A, B. Erv-C and C. double mutant of Erv-C. Lower panels of Figures A, B and C are corresponding schematic representations of substrate interactions as observed in the lowest energy model of each of the enzyme-substrate complexes. D. Root mean square deviations (rmsd) in Å of the substrate for 100 poses as mentioned above. The rmsd of the same in the entire 1 ns trajectory has been shown in the inset figure. E. The rmsd in Å of the main chain of the three enzymes for the minimized initial 100 conformations. F. The side-chain torsion angles of Tyr67 in Erv-A and in the double mutant of Erv-C for 100 conformations.
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pone-0062619-g007: Substrate docked at the active site cleft.Overlay of 100 poses (generated after minimization of initial 100 conformations of MD trajectory) of the substrate (N-benzoyl-Phe-Val-Arg-↓-pNA) docked at the active sites of A. Erv-A, B. Erv-C and C. double mutant of Erv-C. Lower panels of Figures A, B and C are corresponding schematic representations of substrate interactions as observed in the lowest energy model of each of the enzyme-substrate complexes. D. Root mean square deviations (rmsd) in Å of the substrate for 100 poses as mentioned above. The rmsd of the same in the entire 1 ns trajectory has been shown in the inset figure. E. The rmsd in Å of the main chain of the three enzymes for the minimized initial 100 conformations. F. The side-chain torsion angles of Tyr67 in Erv-A and in the double mutant of Erv-C for 100 conformations.

Mentions: Substrate residues flanking the scissile bond are major determinants for site-specific cleavage by proteases [22]. They are generally defined as Pn–P1 (unprimed) for the residues N-terminal to the scissile peptide bond and P1′–Pn′ (primed) for the residues C-terminal to the scissile bond [23]. To understand the binding and hydrolysis of peptides by Erv-A, Erv-C and the double mutant (S32T/A67Y) of Erv-C, we simulated the binding of the substrate N-benzoyl(P4)-Phe(P3)-Val(P2)-Arg(P1)-↓-pNA(P1′) in the active sites of these proteases (Fig. 7A, 7B, and 7C). The reason for choosing this substrate was that it had been used for our experimental enzyme kinetic analysis so that the observations from the modeling studies can be correlated with the enzyme kinetic data. The substrate binds to Erv-A tightly and all the 100 binding conformers are stable at the catalytic cleft with low root mean square deviation (rmsd) values (Fig. 7A and 7D). In comparison, the substrate, docked in the catalytic cleft of Erv-C, shows conformational flexibility with high rmsd values (Fig. 7B and 7D). For the double mutant, the flexibility of the substrate is restricted compared to Erv-C, however rigidity is not achieved as that of Erv-A (Fig. 7C and 7D). The main-chain rmsd values (Fig. 7E) are low (0.4–0.6 Å) in all the three enzymes indicating structural stability for all of them. The side-chain of Tyr67 of Erv-A orients towards S2–S3 cleft, forming hydrophobic and л-л interactions with Val(P2) and Phe(P3) of the substrate respectively (Fig. 7A). Even though Erv-C double mutant has the same residue at 67, its orientation is different, providing less contribution towards binding of Val(P2) and Phe(P3) of the substrate unlike Erv-A. The orientation of Tyr67 in both the enzymes is consistent in all 100 conformations (Fig. 7F). Thus the presence of Tyr67 is not only important, its orientation also plays a crucial role suggesting that the residue behaves as a hydrophobic lock of the P2/P3 residues of the substrate in the S2/S3 pockets with maximum effect for a particular orientation as is found in Erv-A. It is known that the surface charge is also important for substrate recognition. As is mentioned in the previous section, Erv-C contains a strong electronegative surface near S1 site which pulls the guanidium group of Arg(P1) side-chain of the substrate (Fig. 7B). Because of the presence of negative surface charge near S1 subsite along with absence of Tyr67 at S2/S3 subsite in Erv-C, the substrate can not fix itself at the catalytic cleft and the position of the carbonyl O atom of the scissile bond is flexible compared to that of Erv-A reducing the propensity for a nucleophillic attack by the catalytic Cysteine. However, due to mutations in Erv-C double mutant the intensity of negative charge near S1 site is reduced and Arg(P1) side-chain points outward to the solvent. Moreover presence of Tyr67 in the double mutant, whatever is the side chain orientation, has some hydrophobic effect on binding of Val(P2) and Phe(P3) which reduces the flexibility of the substrate at the catalytic cleft. This effect finally helps in positioning the scissile bond of the substrate in proper position between the catalytic dyad and the carbonyl oxygen atom of the scissile bond in the oxyanionic hole of the enzyme, enhancing propensity of nucleophillic attack by the catalytic Cysteine more than wild-type Erv-C. The calculated interaction energies between enzymes and substrate of the complexes (Erv-C: −168.07 kcal/mol, Erv-C double mutant: −79.97 kcal/mol and Erv-A: −46.63 kcal/mol) do not correspond to the experimental catalytic efficiencies (kcat/Km). The electrostatic interaction between P1(Arg) and Erv-C surface charge contribute to the interaction energy, though this interaction do not allow in taking proper position of the P1′-P1 scissile peptide bond for nucleophillic attack by the enzyme. This may be a possible reason for lower catalytic efficiency of Erv-C inspite of higher interaction energy between the enzyme and the substrate.


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

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

Substrate docked at the active site cleft.Overlay of 100 poses (generated after minimization of initial 100 conformations of MD trajectory) of the substrate (N-benzoyl-Phe-Val-Arg-↓-pNA) docked at the active sites of A. Erv-A, B. Erv-C and C. double mutant of Erv-C. Lower panels of Figures A, B and C are corresponding schematic representations of substrate interactions as observed in the lowest energy model of each of the enzyme-substrate complexes. D. Root mean square deviations (rmsd) in Å of the substrate for 100 poses as mentioned above. The rmsd of the same in the entire 1 ns trajectory has been shown in the inset figure. E. The rmsd in Å of the main chain of the three enzymes for the minimized initial 100 conformations. F. The side-chain torsion angles of Tyr67 in Erv-A and in the double mutant of Erv-C for 100 conformations.
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pone-0062619-g007: Substrate docked at the active site cleft.Overlay of 100 poses (generated after minimization of initial 100 conformations of MD trajectory) of the substrate (N-benzoyl-Phe-Val-Arg-↓-pNA) docked at the active sites of A. Erv-A, B. Erv-C and C. double mutant of Erv-C. Lower panels of Figures A, B and C are corresponding schematic representations of substrate interactions as observed in the lowest energy model of each of the enzyme-substrate complexes. D. Root mean square deviations (rmsd) in Å of the substrate for 100 poses as mentioned above. The rmsd of the same in the entire 1 ns trajectory has been shown in the inset figure. E. The rmsd in Å of the main chain of the three enzymes for the minimized initial 100 conformations. F. The side-chain torsion angles of Tyr67 in Erv-A and in the double mutant of Erv-C for 100 conformations.
Mentions: Substrate residues flanking the scissile bond are major determinants for site-specific cleavage by proteases [22]. They are generally defined as Pn–P1 (unprimed) for the residues N-terminal to the scissile peptide bond and P1′–Pn′ (primed) for the residues C-terminal to the scissile bond [23]. To understand the binding and hydrolysis of peptides by Erv-A, Erv-C and the double mutant (S32T/A67Y) of Erv-C, we simulated the binding of the substrate N-benzoyl(P4)-Phe(P3)-Val(P2)-Arg(P1)-↓-pNA(P1′) in the active sites of these proteases (Fig. 7A, 7B, and 7C). The reason for choosing this substrate was that it had been used for our experimental enzyme kinetic analysis so that the observations from the modeling studies can be correlated with the enzyme kinetic data. The substrate binds to Erv-A tightly and all the 100 binding conformers are stable at the catalytic cleft with low root mean square deviation (rmsd) values (Fig. 7A and 7D). In comparison, the substrate, docked in the catalytic cleft of Erv-C, shows conformational flexibility with high rmsd values (Fig. 7B and 7D). For the double mutant, the flexibility of the substrate is restricted compared to Erv-C, however rigidity is not achieved as that of Erv-A (Fig. 7C and 7D). The main-chain rmsd values (Fig. 7E) are low (0.4–0.6 Å) in all the three enzymes indicating structural stability for all of them. The side-chain of Tyr67 of Erv-A orients towards S2–S3 cleft, forming hydrophobic and л-л interactions with Val(P2) and Phe(P3) of the substrate respectively (Fig. 7A). Even though Erv-C double mutant has the same residue at 67, its orientation is different, providing less contribution towards binding of Val(P2) and Phe(P3) of the substrate unlike Erv-A. The orientation of Tyr67 in both the enzymes is consistent in all 100 conformations (Fig. 7F). Thus the presence of Tyr67 is not only important, its orientation also plays a crucial role suggesting that the residue behaves as a hydrophobic lock of the P2/P3 residues of the substrate in the S2/S3 pockets with maximum effect for a particular orientation as is found in Erv-A. It is known that the surface charge is also important for substrate recognition. As is mentioned in the previous section, Erv-C contains a strong electronegative surface near S1 site which pulls the guanidium group of Arg(P1) side-chain of the substrate (Fig. 7B). Because of the presence of negative surface charge near S1 subsite along with absence of Tyr67 at S2/S3 subsite in Erv-C, the substrate can not fix itself at the catalytic cleft and the position of the carbonyl O atom of the scissile bond is flexible compared to that of Erv-A reducing the propensity for a nucleophillic attack by the catalytic Cysteine. However, due to mutations in Erv-C double mutant the intensity of negative charge near S1 site is reduced and Arg(P1) side-chain points outward to the solvent. Moreover presence of Tyr67 in the double mutant, whatever is the side chain orientation, has some hydrophobic effect on binding of Val(P2) and Phe(P3) which reduces the flexibility of the substrate at the catalytic cleft. This effect finally helps in positioning the scissile bond of the substrate in proper position between the catalytic dyad and the carbonyl oxygen atom of the scissile bond in the oxyanionic hole of the enzyme, enhancing propensity of nucleophillic attack by the catalytic Cysteine more than wild-type Erv-C. The calculated interaction energies between enzymes and substrate of the complexes (Erv-C: −168.07 kcal/mol, Erv-C double mutant: −79.97 kcal/mol and Erv-A: −46.63 kcal/mol) do not correspond to the experimental catalytic efficiencies (kcat/Km). The electrostatic interaction between P1(Arg) and Erv-C surface charge contribute to the interaction energy, though this interaction do not allow in taking proper position of the P1′-P1 scissile peptide bond for nucleophillic attack by the enzyme. This may be a possible reason for lower catalytic efficiency of Erv-C inspite of higher interaction energy between the enzyme and the substrate.

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