Biochemical characterization of the transcriptional regulator BzdR from Azoarcus sp. CIB

Gonzalo Durante-Rodríguez, J. Andrés Valderrama, José Miguel Mancheño, Germán Rivas, Carlos Alfonso, Ernesto Arias-Palomo, Oscar Llorca, José Luis García, Eduardo Díaz, Manuel Carmona

Research output: Contribution to journalArticlepeer-review

Abstract

The BzdR transcriptional regulator that controls the PN promoter responsible for the anaerobic catabolism of benzoate in Azoarcus sp. CIB constitutes the prototype of a new subfamily of transcriptional regulators. Here, we provide some insights about the functional-structural relationships of the BzdR protein. Analytical ultracentrifugation studies revealed that BzdR is homodimeric in solution. An electron microscopy three-dimensional reconstruction of the BzdR dimer has been obtained, and the predicted structures of the respective N- and C-terminal domains of each BzdR monomer could be fitted into such a reconstruction. Gel retardation and ultracentrifugation experiments have shown that the binding of BzdR to its cognate promoter is cooperative. Different biochemical approaches revealed that the effector molecule benzoyl-CoA induces conformational changes in BzdR without affecting its oligomeric state. The BzdR-dependent inhibition of the PN promoter and its activation in the presence of benzoyl-CoA have been established by in vitro transcription assays. The monomeric BzdR4 and BzdR5 mutant regulators revealed that dimerization of BzdR isessential for DNA binding. Remarkably, a BzdRΔL protein lacking the linker region connecting the N- and C-terminal domains of BzdR is also dimeric and behaves as a super-repressor of the PN promoter. These data suggest that the linker region of BzdR is not essential for protein dimerization, but rather it is required to transfer the conformational changes induced by the benzoyl-CoA to the DNA binding domain leading to the release of the repressor. A model of action of the BzdR regulator has been proposed.

Original languageEnglish (US)
Pages (from-to)35694-35705
Number of pages12
JournalJournal of Biological Chemistry
Volume285
Issue number46
DOIs
StatePublished - Nov 12 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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