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Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis
BACKGROUND: YedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228395/ https://www.ncbi.nlm.nih.gov/pubmed/24180491 http://dx.doi.org/10.1186/1471-2091-14-28 |
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author | Sabaty, Monique Grosse, Sandrine Adryanczyk, Geraldine Boiry, Séverine Biaso, Frédéric Arnoux, Pascal Pignol, David |
author_facet | Sabaty, Monique Grosse, Sandrine Adryanczyk, Geraldine Boiry, Séverine Biaso, Frédéric Arnoux, Pascal Pignol, David |
author_sort | Sabaty, Monique |
collection | PubMed |
description | BACKGROUND: YedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity. RESULTS: In this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme’s reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher. CONCLUSION: Our study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme. |
format | Online Article Text |
id | pubmed-4228395 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-42283952014-11-13 Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis Sabaty, Monique Grosse, Sandrine Adryanczyk, Geraldine Boiry, Séverine Biaso, Frédéric Arnoux, Pascal Pignol, David BMC Biochem Research Article BACKGROUND: YedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity. RESULTS: In this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme’s reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher. CONCLUSION: Our study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme. BioMed Central 2013-11-01 /pmc/articles/PMC4228395/ /pubmed/24180491 http://dx.doi.org/10.1186/1471-2091-14-28 Text en Copyright © 2013 Sabaty et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 Sabaty, Monique Grosse, Sandrine Adryanczyk, Geraldine Boiry, Séverine Biaso, Frédéric Arnoux, Pascal Pignol, David Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis |
title | Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis |
title_full | Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis |
title_fullStr | Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis |
title_full_unstemmed | Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis |
title_short | Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis |
title_sort | detrimental effect of the 6 his c-terminal tag on yedy enzymatic activity and influence of the tat signal sequence on yedy synthesis |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228395/ https://www.ncbi.nlm.nih.gov/pubmed/24180491 http://dx.doi.org/10.1186/1471-2091-14-28 |
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