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.
Highlights
Proteases or proteolytic enzymes regulate various important biological processes in a living cell and they are extensively studied and well characterized [1]
S32T and S32T/A67Y could be activated by giving a short heat shock for 30 sec at 60uC while for A67Y, an incubation of 10 min at 60uC is required to get maximum activity
Catalytic activity is enhanced in two single mutants (S32T and A67Y) and in a double mutant (S32T/A67Y) of a thermostable papain-like cysteine protease Erv-C without compromising its thermal stability by using a structure-based sitedirected mutagenesis approach
Summary
Proteases or proteolytic enzymes regulate various important biological processes in a living cell and they are extensively studied and well characterized [1]. The existing commercially available plant cysteine proteases have a high degree of proteolytic activity, the need for new proteases with more appealing physicochemical properties for industry are emerging [8] In this context, we can mention that higher stability of industrial enzymes is generally considered as an economic advantage because of reduced enzyme turnover. There are two general strategies for protein engineering: 1) directed evolution by randomly mutating the gene and subsequent selection of the mutant(s) with desired properties and 2) rational design in which targeted mutations are carried out in the gene, guided by structural analyses using X-ray crystallography and molecular modelling [8,11,12]. This latter approach has been proven to be straightforward for generating desired properties in enzymes, if three dimensional (3D) structures of the concerned enzymes are available
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