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 104-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.
Highlights
While halide chemistry is a mainstay of modern chemical synthesis and products, the presence of halide atoms within biological molecules is a relatively rare occurrence [1,2]
The high toxicity, persistence and bioaccumulation of chlorinated molecules such as polychlorinated biphenyls (PCBs) or dioxins is of particular concern, and pollution of the environment and/or food chain by these compounds is often exacerbated by detection problems [3,4]
Organohaliderespiring bacteria have been shown to use a range of chlorinated molecules as terminal electron acceptors, and these organisms as well as the molecular components underpinning this process have the potential for future applications in bioremediation/biosensing of PCB/dioxin-type compounds [5]
Summary
While halide chemistry is a mainstay of modern chemical synthesis and products, the presence of halide atoms within biological molecules is a relatively rare occurrence [1,2]. To confirm the apparent Kd value observed reflects inherent CprK affinity for the effector molecule, we measured the increase in fluorescence levels for a range of aromatic compounds. The 40-fold difference between apparent Kd values for HPA and o-3-fluoro-4-hydroxyphenyl acetic acid (o-FPA) is noteworthy, as H and F have a very similar atomic radius but have distinct electronegativity This supports the pKa interrogation hypothesis for the mechanism of CprK, as the pKa between HPA and o-FPA is affected by the F-substituent. To explore the relationship between atomic radius of the PA o-functional group and CprK affinity in the absence of significant pKa effects, we tested a range of p-nitrophenolic compounds In this series, the electron-withdrawing effects of the p-nitro substituent largely determine the phenol pKa. (c) Both atomic radius and electronegativity of the phenylacetic acid ortho-functional group determine
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