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. 2013 May 3;8(5):e62619.
doi: 10.1371/journal.pone.0062619. Print 2013.

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

Sruti Dutta  1 , Jiban Kanti Dattagupta, Sampa Biswas
Affiliations

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

Sruti Dutta et al. PLoS One. .

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 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.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. Time course of activation of pro-enzymes to the mature and active form of the wild-type and the mutants of Erv-C.
Aliquots of purified pro-enzymes (10–20 µg) were treated for activation for 0 to 50 minutes to convert into their respective mature forms and the percentage of residual enzyme activities were determined with respect to the maximum activity using an azocasein assay, as described in Materials and methods.
Figure 4
Figure 4. 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.
Figure 5
Figure 5. Analyses of thermal stability of the wild-type and the mutants of Erv-C.
A. Determination of optimum temperature of activity (T opt) 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 temperatures as described in Materials and methods. B. Effect of temperature on activity of the wild type and the mutants of Erv-C. Each purified pro-enzyme (10–20 µg) was treated for 10 min at different temperatures followed by activation of the pro-enzymes to their respective mature forms. The percentage residual enzyme activities (at each temperature) were determined with respect to the maximum activity using an azocasein assay. Each data point is an average of three independent experiments having similar values for both the graphs.
Figure 6
Figure 6. 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.
Figure 7
Figure 7. 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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References

    1. Grudkowska M, Zagdanska B (2004) Multifunctional role of plant cysteine proteinases. Acta Biochim Pol 51: 609–624. - PubMed
    1. Barrett AJ, Rawlings ND, Woessner JF (1998) Handbook of proteolytic enzymes. second ed. London: Academic Press.
    1. Baker PJ, Numata K (2012) Chemoenzymatic synthesis of 1 Poly (L-alanine) in aqueous environment. Biomacromolecules 13: 947–951. - PubMed
    1. Tuck SA, Berkas VL, Beam S, Anderson JC (2012) Bronchoscopic thermal vapour ablation in a canine model of emphysema. International journal of COPD 7: 21–31. - PMC - PubMed
    1. Behnke JM, Buttle DJ, Gillian S, Lowe A, Duce IR (2008) Developing novel anthelmintics from plant cysteine proteinases. Parasites & Vectors 1: 29. - PMC - PubMed

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Grants and funding

In addition to institute funding from Department of Atomic Energy (DAE), this work was supported by Council of Scientific and Industrial Research (CSIR), (21/0653/06/EMR-II) and Department of Biotechnology (DBT) (BT/PR13895/BRB/10/789/2010), Govt. of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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