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A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site
BACKGROUND: The Escherichia coli enzyme tryptophanase (TnaA) converts tryptophan to indole, which triggers physiological changes and regulates interactions between bacteria and their mammalian hosts. Tryptophanase production is induced by external tryptophan, but the activity of TnaA is also regulat...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323232/ https://www.ncbi.nlm.nih.gov/pubmed/25650045 http://dx.doi.org/10.1186/s12866-015-0346-3 |
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author | Li, Gang Young, Kevin D |
author_facet | Li, Gang Young, Kevin D |
author_sort | Li, Gang |
collection | PubMed |
description | BACKGROUND: The Escherichia coli enzyme tryptophanase (TnaA) converts tryptophan to indole, which triggers physiological changes and regulates interactions between bacteria and their mammalian hosts. Tryptophanase production is induced by external tryptophan, but the activity of TnaA is also regulated by other, more poorly understood mechanisms. For example, the enzyme accumulates as a spherical inclusion (focus) at midcell or at one pole, but how or why this localization occurs is unknown. RESULTS: TnaA activity is low when the protein forms foci during mid-logarithmic growth but its activity increases as the protein becomes more diffuse, suggesting that foci may represent clusters of inactive (or less active) enzyme. To determine what protein characteristics might mediate these localization effects, we constructed 42 TnaA variants: 6 truncated forms and 36 missense mutants in which different combinations of 83 surface-exposed residues were converted to alanine. A truncated TnaA protein containing only domains D1 and D3 (D1D3) localized to the pole. Mutations affecting the D1D3-to-D1D3 interface did not affect polar localization of D1D3 but did delay assembly of wild type TnaA foci. In contrast, alterations to the D1D3-to-D2 domain interface produced diffuse localization of the D1D3 variant but did not affect the wild type protein. Altering several surface-exposed residues decreased TnaA activity, implying that tetramer assembly may depend on interactions involving these sites. Interestingly, changing any of three amino acids at the base of a loop near the catalytic pocket decreased TnaA activity and caused it to form elongated ovoid foci in vivo, indicating that the alterations affect focus formation and may regulate how frequently tryptophan reaches the active site. CONCLUSIONS: The results suggest that TnaA activity is regulated by subcellular localization and by a loop-associated occlusion of its active site. Equally important, these new TnaA variants are immediately available to the research community and should be useful for investigating how tryptophanase is localized and assembled, how substrate accesses its active site, the functional role of acetylation, and other structural and functional questions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12866-015-0346-3) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4323232 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-43232322015-02-11 A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site Li, Gang Young, Kevin D BMC Microbiol Research Article BACKGROUND: The Escherichia coli enzyme tryptophanase (TnaA) converts tryptophan to indole, which triggers physiological changes and regulates interactions between bacteria and their mammalian hosts. Tryptophanase production is induced by external tryptophan, but the activity of TnaA is also regulated by other, more poorly understood mechanisms. For example, the enzyme accumulates as a spherical inclusion (focus) at midcell or at one pole, but how or why this localization occurs is unknown. RESULTS: TnaA activity is low when the protein forms foci during mid-logarithmic growth but its activity increases as the protein becomes more diffuse, suggesting that foci may represent clusters of inactive (or less active) enzyme. To determine what protein characteristics might mediate these localization effects, we constructed 42 TnaA variants: 6 truncated forms and 36 missense mutants in which different combinations of 83 surface-exposed residues were converted to alanine. A truncated TnaA protein containing only domains D1 and D3 (D1D3) localized to the pole. Mutations affecting the D1D3-to-D1D3 interface did not affect polar localization of D1D3 but did delay assembly of wild type TnaA foci. In contrast, alterations to the D1D3-to-D2 domain interface produced diffuse localization of the D1D3 variant but did not affect the wild type protein. Altering several surface-exposed residues decreased TnaA activity, implying that tetramer assembly may depend on interactions involving these sites. Interestingly, changing any of three amino acids at the base of a loop near the catalytic pocket decreased TnaA activity and caused it to form elongated ovoid foci in vivo, indicating that the alterations affect focus formation and may regulate how frequently tryptophan reaches the active site. CONCLUSIONS: The results suggest that TnaA activity is regulated by subcellular localization and by a loop-associated occlusion of its active site. Equally important, these new TnaA variants are immediately available to the research community and should be useful for investigating how tryptophanase is localized and assembled, how substrate accesses its active site, the functional role of acetylation, and other structural and functional questions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12866-015-0346-3) contains supplementary material, which is available to authorized users. BioMed Central 2015-02-04 /pmc/articles/PMC4323232/ /pubmed/25650045 http://dx.doi.org/10.1186/s12866-015-0346-3 Text en © Li and Young; licensee BioMed Central. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Article Li, Gang Young, Kevin D A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site |
title | A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site |
title_full | A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site |
title_fullStr | A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site |
title_full_unstemmed | A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site |
title_short | A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site |
title_sort | new suite of tnaa mutants suggests that escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323232/ https://www.ncbi.nlm.nih.gov/pubmed/25650045 http://dx.doi.org/10.1186/s12866-015-0346-3 |
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