The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase
ABSTRACT 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....
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American Society for Microbiology
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Online Access: | http://dx.doi.org/10.1128/jb.187.17.6197-6205.2005 https://journals.asm.org/doi/pdf/10.1128/JB.187.17.6197-6205.2005 |
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crasmicro:10.1128/jb.187.17.6197-6205.2005 2024-09-15T17:45:57+00:00 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 http://dx.doi.org/10.1128/jb.187.17.6197-6205.2005 https://journals.asm.org/doi/pdf/10.1128/JB.187.17.6197-6205.2005 en eng American Society for Microbiology https://journals.asm.org/non-commercial-tdm-license Journal of Bacteriology volume 187, issue 17, page 6197-6205 ISSN 0021-9193 1098-5530 journal-article 2005 crasmicro https://doi.org/10.1128/jb.187.17.6197-6205.2005 2024-08-12T04:06:30Z ABSTRACT 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 Ca 2+ 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. Article in Journal/Newspaper Antarc* Antarctic ASM Journals (American Society for Microbiology) Journal of Bacteriology 187 17 6197 6205 |
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Open Polar |
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ASM Journals (American Society for Microbiology) |
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crasmicro |
language |
English |
description |
ABSTRACT 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 Ca 2+ 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 |
Article in Journal/Newspaper |
author |
Siddiqui, Khawar S. Feller, Georges D'Amico, Salvino Gerday, Charles Giaquinto, Laura Cavicchioli, Ricardo |
spellingShingle |
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 |
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://dx.doi.org/10.1128/jb.187.17.6197-6205.2005 https://journals.asm.org/doi/pdf/10.1128/JB.187.17.6197-6205.2005 |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Journal of Bacteriology volume 187, issue 17, page 6197-6205 ISSN 0021-9193 1098-5530 |
op_rights |
https://journals.asm.org/non-commercial-tdm-license |
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 |
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1810493883026505728 |