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Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

ABSTRACT: Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pK(a) of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14-Cys49' intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9?Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition ?4 and ?4' helices ?4?Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

SUBMITTER: Palm GJ

PROVIDER: S-EPMC3351151 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

Palm Gottfried J GJ Khanh Chi Bui B Waack Paul P Gronau Katrin K Becher Dörte D Albrecht Dirk D Hinrichs Winfried W Read Randy J RJ Antelmann Haike H

Nucleic acids research 20120111 9

Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 ...[more]

PMID: 22238377

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Project description:Reductive dehalogenases are essential enzymes in organohalide respiration and consist of a catalytic subunit A and a membrane protein B, encoded by rdhAB genes. Thirty-two rdhAB genes exist in the genome of Dehalococcoides mccartyi strain CBDB1. To gain a first insight into the regulation of rdh operons, the control of gene expression of two rdhAB genes (cbdbA1453/cbdbA1452 and cbdbA1455/cbdbA1454) by the MarR-type regulator Rdh2R (cbdbA1456) encoded directly upstream was studied using heterologous expression and in vitro studies. Promoter-lacZ reporter fusions were generated and integrated into the genome of the Escherichia coli host. The lacZ reporter activities of both rdhA promoters decreased upon transformation of the cells with a plasmid carrying the rdh2R gene, suggesting that Rdh2R acts as repressor, whereas the lacZ reporter activity of the rdh2R promoter was not affected. The transcriptional start sites of both rdhA genes in strain CBDB1 and/or the heterologous host mapped to a conserved direct repeat with 11- to 13-bp half-sites. DNase I footprinting revealed binding of Rdh2R to a ∼30-bp sequence covering the complete direct repeat in both promoters, including the transcriptional start sites. Equilibrium sedimentation ultracentrifugation revealed that Rdh2R binds as tetramer to the direct-repeat motif of the rdhA (cbdbA1455) promoter. Using electrophoretic mobility shift assays, a similar binding affinity was found for both rdhA promoters. In the presence of only one half-site of the direct repeat, the interaction was strongly reduced, suggesting a positive cooperativity of binding, for which unusual short palindromes within the direct-repeat half-sites might play an important role.ImportanceDehalococcoides mccartyi strains are obligate anaerobes that grow by organohalide respiration. They have an important bioremediation potential because they are capable of reducing a multitude of halogenated compounds to less toxic products. We are now beginning to understand how these organisms make use of this large catabolic potential, whereby D. mccartyi expresses dehalogenases in a compound-specific fashion. MarR-type regulators are often encoded in the vicinity of reductive dehalogenase genes. In this study, we made use of heterologous expression and in vitro studies to demonstrate that the MarR-type transcription factor Rdh2R acts as a negative regulator. We identify its binding site on the DNA, which suggests a mechanism by which it controls the expression of two adjacent reductive dehalogenase operons.

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Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

Publications

Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

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