Effects of replacing active site residues in a cold‐active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli
Alkaline phosphatase (AP) from a North Atlantic marine Vibrio bacterium was previously characterized as being kinetically cold‐adapted. It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors. There are three metal‐binding...
Published in: | The FEBS Journal |
---|---|
Main Authors: | , |
Format: | Article in Journal/Newspaper |
Language: | English |
Published: |
Wiley
2007
|
Subjects: | |
Online Access: | http://dx.doi.org/10.1111/j.1742-4658.2007.06182.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1742-4658.2007.06182.x https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1742-4658.2007.06182.x |
id |
crwiley:10.1111/j.1742-4658.2007.06182.x |
---|---|
record_format |
openpolar |
spelling |
crwiley:10.1111/j.1742-4658.2007.06182.x 2024-04-28T08:30:59+00:00 Effects of replacing active site residues in a cold‐active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli Gudjónsdóttir, Katrín Ásgeirsson, Bjarni 2007 http://dx.doi.org/10.1111/j.1742-4658.2007.06182.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1742-4658.2007.06182.x https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1742-4658.2007.06182.x en eng Wiley http://onlinelibrary.wiley.com/termsAndConditions#vor The FEBS Journal volume 275, issue 1, page 117-127 ISSN 1742-464X 1742-4658 Cell Biology Molecular Biology Biochemistry journal-article 2007 crwiley https://doi.org/10.1111/j.1742-4658.2007.06182.x 2024-04-02T08:46:03Z Alkaline phosphatase (AP) from a North Atlantic marine Vibrio bacterium was previously characterized as being kinetically cold‐adapted. It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors. There are three metal‐binding sites in the active site of APs, and all three metal ions participate in catalysis. The amino acid residues that bind the two zinc ions most commonly present are conserved in all known APs. In contrast, two of the residues that bind the third metal ion (numbered 153 and 328 in Escherichia coli AP) are different in various APs. This may explain their different catalytic efficiencies, as the Mg 2+ most often present there is important for both structural stability and the reaction mechanism. We have mutated these key residues to the corresponding residues in E. coli AP to obtain the double mutant Asp116/Lys274, and both single mutants. All these mutants displayed reduced substrate affinity and lower overall reaction rates. The Lys274 and Asp116/Lys274 mutants also displayed an increase in global heat stability, which may be due to the formation of a stabilizing salt bridge. Overall, the results show that a single amino acid substitution in the active site is sufficient to alter the structural stability of the cold‐active Vibrio AP both locally and globally, and this influences kinetic properties. Article in Journal/Newspaper North Atlantic Wiley Online Library The FEBS Journal 275 1 117 127 |
institution |
Open Polar |
collection |
Wiley Online Library |
op_collection_id |
crwiley |
language |
English |
topic |
Cell Biology Molecular Biology Biochemistry |
spellingShingle |
Cell Biology Molecular Biology Biochemistry Gudjónsdóttir, Katrín Ásgeirsson, Bjarni Effects of replacing active site residues in a cold‐active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli |
topic_facet |
Cell Biology Molecular Biology Biochemistry |
description |
Alkaline phosphatase (AP) from a North Atlantic marine Vibrio bacterium was previously characterized as being kinetically cold‐adapted. It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors. There are three metal‐binding sites in the active site of APs, and all three metal ions participate in catalysis. The amino acid residues that bind the two zinc ions most commonly present are conserved in all known APs. In contrast, two of the residues that bind the third metal ion (numbered 153 and 328 in Escherichia coli AP) are different in various APs. This may explain their different catalytic efficiencies, as the Mg 2+ most often present there is important for both structural stability and the reaction mechanism. We have mutated these key residues to the corresponding residues in E. coli AP to obtain the double mutant Asp116/Lys274, and both single mutants. All these mutants displayed reduced substrate affinity and lower overall reaction rates. The Lys274 and Asp116/Lys274 mutants also displayed an increase in global heat stability, which may be due to the formation of a stabilizing salt bridge. Overall, the results show that a single amino acid substitution in the active site is sufficient to alter the structural stability of the cold‐active Vibrio AP both locally and globally, and this influences kinetic properties. |
format |
Article in Journal/Newspaper |
author |
Gudjónsdóttir, Katrín Ásgeirsson, Bjarni |
author_facet |
Gudjónsdóttir, Katrín Ásgeirsson, Bjarni |
author_sort |
Gudjónsdóttir, Katrín |
title |
Effects of replacing active site residues in a cold‐active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli |
title_short |
Effects of replacing active site residues in a cold‐active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli |
title_full |
Effects of replacing active site residues in a cold‐active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli |
title_fullStr |
Effects of replacing active site residues in a cold‐active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli |
title_full_unstemmed |
Effects of replacing active site residues in a cold‐active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli |
title_sort |
effects of replacing active site residues in a cold‐active alkaline phosphatase with those found in its mesophilic counterpart from escherichia coli |
publisher |
Wiley |
publishDate |
2007 |
url |
http://dx.doi.org/10.1111/j.1742-4658.2007.06182.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1742-4658.2007.06182.x https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1742-4658.2007.06182.x |
genre |
North Atlantic |
genre_facet |
North Atlantic |
op_source |
The FEBS Journal volume 275, issue 1, page 117-127 ISSN 1742-464X 1742-4658 |
op_rights |
http://onlinelibrary.wiley.com/termsAndConditions#vor |
op_doi |
https://doi.org/10.1111/j.1742-4658.2007.06182.x |
container_title |
The FEBS Journal |
container_volume |
275 |
container_issue |
1 |
container_start_page |
117 |
op_container_end_page |
127 |
_version_ |
1797588672388792320 |