Engineering the properties of a cold active enzyme through rational redesign of the active site
In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side‐chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 an...
Published in: | European Journal of Biochemistry |
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Language: | English |
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Online Access: | http://dx.doi.org/10.1046/j.0014-2956.2001.02432.x http://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.0014-2956.2001.02432.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.0014-2956.2001.02432.x https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.0014-2956.2001.02432.x |
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crwiley:10.1046/j.0014-2956.2001.02432.x 2024-06-02T07:57:38+00:00 Engineering the properties of a cold active enzyme through rational redesign of the active site Tsigos, Iason Mavromatis, Konstantinos Tzanodaskalaki, Maria Pozidis, Charalambos Kokkinidis, Michael Bouriotis, Vassilis 2001 http://dx.doi.org/10.1046/j.0014-2956.2001.02432.x http://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.0014-2956.2001.02432.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.0014-2956.2001.02432.x https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.0014-2956.2001.02432.x en eng Wiley http://onlinelibrary.wiley.com/termsAndConditions#vor European Journal of Biochemistry volume 268, issue 19, page 5074-5080 ISSN 0014-2956 1432-1033 journal-article 2001 crwiley https://doi.org/10.1046/j.0014-2956.2001.02432.x 2024-05-03T11:31:19Z In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side‐chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg 2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild‐type in the temperature range 5–25 °C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5–15 °C and a significantly increased activity at 20–25 °C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135→Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild‐type sequence, which may affect active site flexibility through changes in various conformational constraints. Article in Journal/Newspaper Antarc* Antarctic Wiley Online Library Antarctic The Antarctic European Journal of Biochemistry 268 19 5074 5080 |
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Wiley Online Library |
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crwiley |
language |
English |
description |
In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side‐chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg 2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild‐type in the temperature range 5–25 °C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5–15 °C and a significantly increased activity at 20–25 °C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135→Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild‐type sequence, which may affect active site flexibility through changes in various conformational constraints. |
format |
Article in Journal/Newspaper |
author |
Tsigos, Iason Mavromatis, Konstantinos Tzanodaskalaki, Maria Pozidis, Charalambos Kokkinidis, Michael Bouriotis, Vassilis |
spellingShingle |
Tsigos, Iason Mavromatis, Konstantinos Tzanodaskalaki, Maria Pozidis, Charalambos Kokkinidis, Michael Bouriotis, Vassilis Engineering the properties of a cold active enzyme through rational redesign of the active site |
author_facet |
Tsigos, Iason Mavromatis, Konstantinos Tzanodaskalaki, Maria Pozidis, Charalambos Kokkinidis, Michael Bouriotis, Vassilis |
author_sort |
Tsigos, Iason |
title |
Engineering the properties of a cold active enzyme through rational redesign of the active site |
title_short |
Engineering the properties of a cold active enzyme through rational redesign of the active site |
title_full |
Engineering the properties of a cold active enzyme through rational redesign of the active site |
title_fullStr |
Engineering the properties of a cold active enzyme through rational redesign of the active site |
title_full_unstemmed |
Engineering the properties of a cold active enzyme through rational redesign of the active site |
title_sort |
engineering the properties of a cold active enzyme through rational redesign of the active site |
publisher |
Wiley |
publishDate |
2001 |
url |
http://dx.doi.org/10.1046/j.0014-2956.2001.02432.x http://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.0014-2956.2001.02432.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.0014-2956.2001.02432.x https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.0014-2956.2001.02432.x |
geographic |
Antarctic The Antarctic |
geographic_facet |
Antarctic The Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
European Journal of Biochemistry volume 268, issue 19, page 5074-5080 ISSN 0014-2956 1432-1033 |
op_rights |
http://onlinelibrary.wiley.com/termsAndConditions#vor |
op_doi |
https://doi.org/10.1046/j.0014-2956.2001.02432.x |
container_title |
European Journal of Biochemistry |
container_volume |
268 |
container_issue |
19 |
container_start_page |
5074 |
op_container_end_page |
5080 |
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1800740812809568256 |