The role of the GAF and central domains of the transcriptional activator VnfA in Azotobacter vinelandii

VnfA is a transcriptional activator that is required for the expression of the structural genes encoding nitrogenase‐2 in Azotobacter vinelandii. VnfA consists of three domains: an N‐terminal regulatory domain termed GAF, including a Cys‐rich motif; a central domain from the AAA+ family; and a C‐ter...

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Bibliographic Details
Published in:The FEBS journal 2011-09, Vol.278 (18), p.3287-3297
Main Authors: Yoshimitsu, Kyohei, Takatani, Nobuyuki, Miura, Yukio, Watanabe, Yoshihito, Nakajima, Hiroshi
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Amino Acid Sequence
Apoenzymes - chemistry
Apoenzymes - metabolism
Azotobacter vinelandii - metabolism
Azotobacter vinelandii
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Biocatalysis
Catalytic Domain
DNA-Binding Proteins - chemistry
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Genes, Reporter
Helix-Turn-Helix Motifs
Holoenzymes - chemistry
Holoenzymes - metabolism
Iron-Sulfur Proteins - chemistry
Iron-Sulfur Proteins - genetics
Iron-Sulfur Proteins - metabolism
iron‐sulfur cluster
Molecular Sequence Data
Molecular Weight
nitrogenase
NTPase activity
Nucleoside-Triphosphatase - chemistry
Nucleoside-Triphosphatase - genetics
Nucleoside-Triphosphatase - metabolism
Peptide Fragments - chemistry
Peptide Fragments - genetics
Peptide Fragments - metabolism
Protein Interaction Domains and Motifs
Protein Multimerization
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Trans-Activators - chemistry
Trans-Activators - genetics
Trans-Activators - metabolism
Transcription, Genetic
transcriptional regulator
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Summary:VnfA is a transcriptional activator that is required for the expression of the structural genes encoding nitrogenase‐2 in Azotobacter vinelandii. VnfA consists of three domains: an N‐terminal regulatory domain termed GAF, including a Cys‐rich motif; a central domain from the AAA+ family; and a C‐terminal domain for DNA binding. Previously, we reported that transcriptionally active VnfA harboring an Fe‐S cluster (presumably of the 3Fe‐4S type) as a prosthetic group and the Cys‐rich motif were possibly associated with coordination of the Fe‐S cluster. In the present study, we have investigated the roles of the GAF and central domains in the regulatory function of VnfA using truncated variants: ΔN15VnfA and ΔGAFVnfA that lack the N‐terminal 15 residues and whole GAF domain, respectively, and GAFVnfA consisting of only the GAF domain. ΔN15VnfA and ΔGAFVnfA lost the ability to bind the Fe‐S cluster, whereas GAFVnfA was still able to bind to the cluster, consistent with the hypothesis that the Cys‐rich motif is essential for Fe‐S cluster binding. The GAF domain showed an inhibitory effect on the transcriptional activity of VnfA, which was reversed in the presence of the Fe‐S cluster, and reactivated upon disassembly of the cluster. The inhibitory activity of the GAF domain acts on the NTPase activity of the central domain, whereas the binding ability of VnfA to DNA was not significantly affected, when VnfA retains its tetrameric conformation. The results imply that a major pathway, by which VnfA function is regulated, operates via the control of NTPase activity by the GAF domain. Structured digital •  VnfA binds to VnfA by molecular sieving (View Interaction 1, 2) Database 
VnfA has been submitted to the Swiss‐Prot database under accession number: C1DI41. VnfA is a σN‐dependent transcriptional activator required for the gene expression of nitrogenase‐2 in Azotobacter vinelandii. We found that the N‐terminal GAF domain of VnfA, which contains an Fe‐S cluster showed an inhibitory effect on the transcriptional activity of VnfA. The inhibitory effect acted on the NTPase activity of the central domain, depending on assembly and disassembly of the cluster.
ISSN:1742-464X
1742-4658
DOI:10.1111/j.1742-4658.2011.08245.x