Chondroitinase ABC I (ChABC I) has been shown to depolymerize a variety of glycosaminoglycan substrates and promote regeneration of damaged spinal cord. However, to date, intrathecal delivery methods have been suboptimal largely due to enzyme instability which necessitates repeated administration to the injured loci. Among the aromatic amino acids, tyrosine has been shown to be more effective in creation of stable clusters and further stabilize of the proteins. Bioinformatics approaches have been used to examine the effect of an extra aromatic cluster at the surface of ChABC I. In this study two amino acids i.e., Asn806 and Gln810 were mutated to tyrosine and to alanine as negative control. In this way, four variants i.e., N806Y/Q810Y, N806A/Q810Y, N806Y/Q810A and N806A/Q810A were created. The results showed that N806Y/Q810Y mutation improved both activity and thermal stability of the enzyme while Ala substitution reduced the enzyme activity and destabilized it. Structural analysis of mutants showed an increase in intrinsic fluorescence intensity and secondary structure content of N806Y/Q810Y mutant when compared to the wild type enzyme indicating a more rigid structure of this variant. Moreover, the N806Y/Q810Y enzyme displayed a remarkable resistance against trypsin degradation with a half-life (t1/2) of 45.0min versus 32.5min of wild-type. In conclusion, the data revealed that structural features and activity of ChABC I can be improved by introducing appropriate aromatic clusters at the surface of the enzyme.
Submitter: Shu-Ching Ou
Submission Date: Oct. 11, 2018, 12:12 p.m.
|Number of data points||65|
|Proteins||Chondroitin sulfate ABC endolyase|
|Assays/Quantities/Protocols||Experimental Assay: Kinetic Constant: Km ; Experimental Assay: Kinetic Constant: kcat ; Experimental Assay: Structural Parameter: α-Helix ; Experimental Assay: Structural Parameter: β-Structure ; Experimental Assay: Structural Parameter: β−Turn ; Experimental Assay: Structural Parameter: Random. Coil ; Experimental Assay: Half-Life ; Experimental Assay: Remained Activity ; Experimental Assay: Fluorescence Quenching: KSV: Acrylamide ; Experimental Assay: Fluorescence Quenching: KSV: Potassium Iodide ; Derived Quantity: SD of Kinetic Constant: Km ; Derived Quantity: SD of Kinetic Constant: kcat ; Derived Quantity: Kinetic Constant: kcat/Km|
|Libraries||Kinetic Parameters, Structural Parameters and Half-Life|