Cargando…
The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms
The transcriptional regulator CprK controls the expression of the reductive dehalogenase CprA in organohalide-respiring bacteria. Desulfitobacterium hafniense CprA catalyses the reductive dechlorination of the terminal electron acceptor o-chlorophenol acetic acid, generating the phenol acetic acid p...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society
2013
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638464/ https://www.ncbi.nlm.nih.gov/pubmed/23479753 http://dx.doi.org/10.1098/rstb.2012.0323 |
_version_ | 1782475830983655424 |
---|---|
author | Kemp, Laura R. Dunstan, Mark S. Fisher, Karl Warwicker, Jim Leys, David |
author_facet | Kemp, Laura R. Dunstan, Mark S. Fisher, Karl Warwicker, Jim Leys, David |
author_sort | Kemp, Laura R. |
collection | PubMed |
description | The transcriptional regulator CprK controls the expression of the reductive dehalogenase CprA in organohalide-respiring bacteria. Desulfitobacterium hafniense CprA catalyses the reductive dechlorination of the terminal electron acceptor o-chlorophenol acetic acid, generating the phenol acetic acid product. It has been shown that CprK has ability to distinguish between the chlorinated CprA substrate and the de-halogenated end product, with an estimated an estimated 10(4)-fold difference in affinity. Using a green fluorescent protein GFP(UV)-based transcriptional reporter system, we establish that CprK can sense o-chlorophenol acetic acid at the nanomolar level, whereas phenol acetic acid leads to transcriptional activation only when approaching micromolar levels. A structure–activity relationship study, using a range of o-chlorophenol acetic-acid-related compounds and key CprK mutants, combined with pK(a) calculations on the effector binding site, suggests that the sensitive detection of chlorination is achieved through a combination of direct and indirect readout mechanisms. Both the physical presence of the bulky chloride substituent as well as the accompanying electronic effects lowering the inherent phenol pK(a) are required for high affinity. Indeed, transcriptional activation by CprK appears strictly dependent on establishing a phenolate–K133 salt bridge interaction, rather than on the presence of a halogen atom per se. As K133 is strictly conserved within the CprK family, our data suggest that physiological function and future applications in biosensing are probably restricted to phenolic compounds. |
format | Online Article Text |
id | pubmed-3638464 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | The Royal Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-36384642013-04-30 The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms Kemp, Laura R. Dunstan, Mark S. Fisher, Karl Warwicker, Jim Leys, David Philos Trans R Soc Lond B Biol Sci Articles The transcriptional regulator CprK controls the expression of the reductive dehalogenase CprA in organohalide-respiring bacteria. Desulfitobacterium hafniense CprA catalyses the reductive dechlorination of the terminal electron acceptor o-chlorophenol acetic acid, generating the phenol acetic acid product. It has been shown that CprK has ability to distinguish between the chlorinated CprA substrate and the de-halogenated end product, with an estimated an estimated 10(4)-fold difference in affinity. Using a green fluorescent protein GFP(UV)-based transcriptional reporter system, we establish that CprK can sense o-chlorophenol acetic acid at the nanomolar level, whereas phenol acetic acid leads to transcriptional activation only when approaching micromolar levels. A structure–activity relationship study, using a range of o-chlorophenol acetic-acid-related compounds and key CprK mutants, combined with pK(a) calculations on the effector binding site, suggests that the sensitive detection of chlorination is achieved through a combination of direct and indirect readout mechanisms. Both the physical presence of the bulky chloride substituent as well as the accompanying electronic effects lowering the inherent phenol pK(a) are required for high affinity. Indeed, transcriptional activation by CprK appears strictly dependent on establishing a phenolate–K133 salt bridge interaction, rather than on the presence of a halogen atom per se. As K133 is strictly conserved within the CprK family, our data suggest that physiological function and future applications in biosensing are probably restricted to phenolic compounds. The Royal Society 2013-04-19 /pmc/articles/PMC3638464/ /pubmed/23479753 http://dx.doi.org/10.1098/rstb.2012.0323 Text en http://creativecommons.org/licenses/by/3.0/ © 2013 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited. |
spellingShingle | Articles Kemp, Laura R. Dunstan, Mark S. Fisher, Karl Warwicker, Jim Leys, David The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms |
title | The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms |
title_full | The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms |
title_fullStr | The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms |
title_full_unstemmed | The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms |
title_short | The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms |
title_sort | transcriptional regulator cprk detects chlorination by combining direct and indirect readout mechanisms |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638464/ https://www.ncbi.nlm.nih.gov/pubmed/23479753 http://dx.doi.org/10.1098/rstb.2012.0323 |
work_keys_str_mv | AT kemplaurar thetranscriptionalregulatorcprkdetectschlorinationbycombiningdirectandindirectreadoutmechanisms AT dunstanmarks thetranscriptionalregulatorcprkdetectschlorinationbycombiningdirectandindirectreadoutmechanisms AT fisherkarl thetranscriptionalregulatorcprkdetectschlorinationbycombiningdirectandindirectreadoutmechanisms AT warwickerjim thetranscriptionalregulatorcprkdetectschlorinationbycombiningdirectandindirectreadoutmechanisms AT leysdavid thetranscriptionalregulatorcprkdetectschlorinationbycombiningdirectandindirectreadoutmechanisms AT kemplaurar transcriptionalregulatorcprkdetectschlorinationbycombiningdirectandindirectreadoutmechanisms AT dunstanmarks transcriptionalregulatorcprkdetectschlorinationbycombiningdirectandindirectreadoutmechanisms AT fisherkarl transcriptionalregulatorcprkdetectschlorinationbycombiningdirectandindirectreadoutmechanisms AT warwickerjim transcriptionalregulatorcprkdetectschlorinationbycombiningdirectandindirectreadoutmechanisms AT leysdavid transcriptionalregulatorcprkdetectschlorinationbycombiningdirectandindirectreadoutmechanisms |