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

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 an...

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Main Authors: Hasnain, Hussain, Chong, Nikson Fatt Ming
Format: Article
Language:English
Published: Turkish Biochemistry Society 2017
Subjects:
Q Science (General)
Online Access:http://ir.unimas.my/id/eprint/17816/2/Implications.pdf
http://ir.unimas.my/id/eprint/17816/
https://www.scopus.com/inward/record.uri?eid=2-s2.0-85020449276&doi=10.1515%2ftjb-2016-0169&partnerID=40&md5=57e0bfc701756f4ce130b0bfa64917ff
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spelling my.unimas.ir.178162022-06-21T01:45:01Z http://ir.unimas.my/id/eprint/17816/ Implications of stisa2 catalytic residue restoration through site directed mutagenesis [Bölgeye yönlendirilmiş mutajenez ile stisa2 katalitik amino asit restorasyonunun olasi sonuçlari] Hasnain, Hussain Chong, Nikson Fatt Ming Q Science (General) 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. Turkish Biochemistry Society 2017 Article PeerReviewed text en http://ir.unimas.my/id/eprint/17816/2/Implications.pdf 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 https://www.scopus.com/inward/record.uri?eid=2-s2.0-85020449276&doi=10.1515%2ftjb-2016-0169&partnerID=40&md5=57e0bfc701756f4ce130b0bfa64917ff DOI: 10.1515/tjb-2016-0169
institution Universiti Malaysia Sarawak
building Centre for Academic Information Services (CAIS)
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaysia Sarawak
content_source UNIMAS Institutional Repository
url_provider http://ir.unimas.my/
language English
topic Q Science (General)
spellingShingle Q Science (General)
Hasnain, Hussain
Chong, Nikson Fatt Ming
Implications of stisa2 catalytic residue restoration through site directed mutagenesis [Bölgeye yönlendirilmiş mutajenez ile stisa2 katalitik amino asit restorasyonunun olasi sonuçlari]
description 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.
format Article
author Hasnain, Hussain
Chong, Nikson Fatt Ming
author_facet Hasnain, Hussain
Chong, Nikson Fatt Ming
author_sort Hasnain, Hussain
title Implications of stisa2 catalytic residue restoration through site directed mutagenesis [Bölgeye yönlendirilmiş mutajenez ile stisa2 katalitik amino asit restorasyonunun olasi sonuçlari]
title_short Implications of stisa2 catalytic residue restoration through site directed mutagenesis [Bölgeye yönlendirilmiş mutajenez ile stisa2 katalitik amino asit restorasyonunun olasi sonuçlari]
title_full Implications of stisa2 catalytic residue restoration through site directed mutagenesis [Bölgeye yönlendirilmiş mutajenez ile stisa2 katalitik amino asit restorasyonunun olasi sonuçlari]
title_fullStr Implications of stisa2 catalytic residue restoration through site directed mutagenesis [Bölgeye yönlendirilmiş mutajenez ile stisa2 katalitik amino asit restorasyonunun olasi sonuçlari]
title_full_unstemmed Implications of stisa2 catalytic residue restoration through site directed mutagenesis [Bölgeye yönlendirilmiş mutajenez ile stisa2 katalitik amino asit restorasyonunun olasi sonuçlari]
title_sort implications of stisa2 catalytic residue restoration through site directed mutagenesis [bölgeye yönlendirilmiş mutajenez ile stisa2 katalitik amino asit restorasyonunun olasi sonuçlari]
publisher Turkish Biochemistry Society
publishDate 2017
url http://ir.unimas.my/id/eprint/17816/2/Implications.pdf
http://ir.unimas.my/id/eprint/17816/
https://www.scopus.com/inward/record.uri?eid=2-s2.0-85020449276&doi=10.1515%2ftjb-2016-0169&partnerID=40&md5=57e0bfc701756f4ce130b0bfa64917ff
_version_ 1736838263420747776
score 13.211869

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