Previously, we attempted to improve the thermostability of chondroitinase ABC I by substituting proline in flexible sites and successfully obtained a mutant; E138P, with increased thermostability (Kheirollahi et al., 2017). In this study, we focused on the role of Glu138 in activity and stability of the enzyme using its further mutation to Ala, Lys, and Asp. Moreover, we coupled the two mutations E138P and Q140A, whose stabilizing effects were reported previously, and evaluated their simultaneous effects on activity, stability, and structure of the enzyme. The results indicate that substitution of Glu138 with the above-mentioned amino acids changed kinetic properties of cABC I but did not lead to increased stability. Moreover, replacement of Glu138 with Lys and Asp caused significant structural changes. These findings lead to the tentative conclusion that improvement in thermal stability of E138P variant is due to the stabilizing effect of proline at position 138. In addition, the double variant showed a significant increase in catalytic efficiency, howbeit its kinetic stability decreased. Moreover, structural analysis of the double mutant form revealed that its tertiary and secondary structure content decreased partially, while its structural flexibility increased.
Submitter: Shu-Ching Ou
Submission Date: Feb. 6, 2019, 1:11 p.m.
|Number of data points||75|
|Proteins||Chondroitin sulfate ABC endolyase|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Experimental Assay: Ksv for KI ; Experimental Assay: Ksv for acrylamide ; Experimental Assay: t1/2 at 37 °C ; Experimental Assay: kcat/Km ; Experimental Assay: kcat ; Experimental Assay: Km ; Experimental Assay: Vmax ; Derived Quantity: SD of Ksv for KI ; Derived Quantity: SD of Ksv for acrylamide ; Derived Quantity: SD of kcat ; Derived Quantity: SD of Km ; Derived Quantity: SD of Vmax|
|Libraries||Variants for cABC-I_C4S ; Variants for cABC I|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|99.8||Chondroitin sulfate ABC endolyase||P59807||CABC1_PROVU|