The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase
The cold-active α-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30°C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was...
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ftpubmed:oai:pubmedcentral.nih.gov:1196144 2023-05-15T14:01:31+02:00 The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase Siddiqui, Khawar S. Feller, Georges D'Amico, Salvino Gerday, Charles Giaquinto, Laura Cavicchioli, Ricardo 2005-09 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196144 http://www.ncbi.nlm.nih.gov/pubmed/16109961 https://doi.org/10.1128/JB.187.17.6197-6205.2005 en eng American Society for Microbiology http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196144 http://www.ncbi.nlm.nih.gov/pubmed/16109961 http://dx.doi.org/10.1128/JB.187.17.6197-6205.2005 Copyright © 2005, American Society for Microbiology Enzymes and Proteins Text 2005 ftpubmed https://doi.org/10.1128/JB.187.17.6197-6205.2005 2013-08-30T12:52:26Z The cold-active α-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30°C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3°C to 70°C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12°C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70°C for 1 h in 6.5 M urea. The study has identified fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes. Text Antarc* Antarctic PubMed Central (PMC) Antarctic The Antarctic Journal of Bacteriology 187 17 6197 6205 |
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language |
English |
topic |
Enzymes and Proteins |
spellingShingle |
Enzymes and Proteins Siddiqui, Khawar S. Feller, Georges D'Amico, Salvino Gerday, Charles Giaquinto, Laura Cavicchioli, Ricardo The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase |
topic_facet |
Enzymes and Proteins |
description |
The cold-active α-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30°C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3°C to 70°C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12°C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70°C for 1 h in 6.5 M urea. The study has identified fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes. |
format |
Text |
author |
Siddiqui, Khawar S. Feller, Georges D'Amico, Salvino Gerday, Charles Giaquinto, Laura Cavicchioli, Ricardo |
author_facet |
Siddiqui, Khawar S. Feller, Georges D'Amico, Salvino Gerday, Charles Giaquinto, Laura Cavicchioli, Ricardo |
author_sort |
Siddiqui, Khawar S. |
title |
The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase |
title_short |
The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase |
title_full |
The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase |
title_fullStr |
The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase |
title_full_unstemmed |
The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase |
title_sort |
active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted α-amylase |
publisher |
American Society for Microbiology |
publishDate |
2005 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196144 http://www.ncbi.nlm.nih.gov/pubmed/16109961 https://doi.org/10.1128/JB.187.17.6197-6205.2005 |
geographic |
Antarctic The Antarctic |
geographic_facet |
Antarctic The Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196144 http://www.ncbi.nlm.nih.gov/pubmed/16109961 http://dx.doi.org/10.1128/JB.187.17.6197-6205.2005 |
op_rights |
Copyright © 2005, American Society for Microbiology |
op_doi |
https://doi.org/10.1128/JB.187.17.6197-6205.2005 |
container_title |
Journal of Bacteriology |
container_volume |
187 |
container_issue |
17 |
container_start_page |
6197 |
op_container_end_page |
6205 |
_version_ |
1766271351393353728 |