Establishment of Aromatic Pairs at the Surface of Chondroitinase ABC I: the Effect on Activity and Stability.


Abstract

Removal of chondroitin sulfate glycosaminoglycan (GAG) chains with chondroitinase ABC I (chABC I) in CNS injury models promotes both saxon regeneration and plasticity. It has been suggested that direct interaction between an aromatic pair appears to contribute about - 1.3 kcal/mol to the stability of a folded protein, so introducing an aromatic pair by point mutation might increase the enzyme activity and thermal stability as in the case of mesophilic xylanase, although using this approach destabilized T4 lysozyme. In this study, we used site-directed mutagenesis to investigate the effect of new aromatic pairs on activity and stability of chABC I. We replaced Ile295, Ser581, and Gly730 adjacent to pre-existing aromatic residues with Tyr to obtain new aromatic pairs, i.e., Tyr295/His372, Tyr576/Tyr581, and Tyr623/Tyr730. Results showed that Km values of S581Y and G730Y variants decreased relative to wild-type enzyme while their catalytic efficiency (kcat/Km) increased but I295Y variant was inactive. Also, long-term and thermal stability of the active mutants was decreased. Fluorescence and circular dichroism studies showed that these mutations resulted in a more flexible enzyme structures: a finding which was confirmed by thermal and limited proteolytic studies. In conclusion, the activity of chABC I can be improved by introducing appropriate aromatic pairs at the enzyme surface. This approach did not provide any promising results regarding the enzyme stability.

Submission Details

ID: TiZ6nMYh3

Submitter: Shu-Ching Ou

Submission Date: March 8, 2019, 12:50 p.m.

Version: 1

Publication Details
Shahaboddin ME;Khajeh K;Golestani A,Appl Biochem Biotechnol (2018) Establishment of Aromatic Pairs at the Surface of Chondroitinase ABC I: the Effect on Activity and Stability. PMID:29627885
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1HN0 2003-04-08 1.9 CRYSTAL STRUCTURE OF CHONDROITIN ABC LYASE I FROM PROTEUS VULGARIS AT 1.9 ANGSTROMS RESOLUTION

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.8 Chondroitin sulfate ABC endolyase P59807 CABC1_PROVU