Implications of stisa2 catalytic residue restoration through site directed mutagenesis [Bölgeye yönlendirilmiş mutajenez ile stisa2 katalitik amino asit restorasyonunun olasi sonuçlari]

Hasnain, Hussain and Chong, Nikson Fatt Ming (2017) Implications of stisa2 catalytic residue restoration through site directed mutagenesis [Bölgeye yönlendirilmiş mutajenez ile stisa2 katalitik amino asit restorasyonunun olasi sonuçlari]. Turkish Journal of Biochemistry, 42 (2). pp. 149-160. ISSN 0250-4685

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Abstract

Objective: Restoration of catalytic activity of Isa2 from Solanum tuberosum (Stisa2) through restoration of conserved catalytic residues by site directed mutagenesis. Methods: The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed in E. coli Rosetta 2 under optimized conditions. Evaluation of debranching activity on starch, amylopectin and β-limit dextrin was carried out by measuring the amount of glucose equivalents released using the bicinchoninic acid assay. Results: Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity. Conclusions: These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity. © 2017 Turkish Biochemistry Society. All rights reserved.

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