title: The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms.
abstract: 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 GFPUV-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 pKa 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 pKa 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.
authors.affiliation: Faculty of Life Sciences, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: Laura R
authors.initials: LR
authors.lastname: Kemp
authors.forename: Mark S
authors.initials: MS
authors.lastname: Dunstan
authors.forename: Karl
authors.initials: K
authors.lastname: Fisher
authors.forename: Jim
authors.initials: J
authors.lastname: Warwicker
authors.forename: David
authors.initials: D
authors.lastname: Leys
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D045858:Desulfitobacterium / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054879:Halogenation; D016297:Mutagenesis, Site-Directed; D010648:Phenylacetates / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D010648:Phenylacetates; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; C008070:4-hydroxyphenylacetic acid