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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The NifL regulatory protein controls transcription of nitrogen fixation genes in Azotobacter vinelandii by modulating the activity of the transcriptional activator NifA through direct protein-protein interactions. The ability of NifL to integrate the antagonistic signals of redox and nitrogen status is achieved via the involvement of discrete domains in signalling specific environmental cues. NifL senses the redox status via an FAD co-factor located within the amino-terminal PAS domain and responds to the fixed nitrogen status by interaction with the signal transduction protein GlnK, which binds to the C-terminal GHKL domain of NifL. The GHKL domain binds adenosine nucleotides and is similar to the core catalytic domain of the histidine protein kinases. Binding of ADP to this domain increases the inhibitory activity of NifL and the formation of protein complexes with NifA. This inhibition is antagonised by the binding of 2-oxoglutarate, a key metabolic signal of the carbon status, to the amino-terminal GAF domain of NifA. In this study we have examined the properties of three mutations within conserved residues in the GHKL domain of NifL that impair signal transduction. All three mutations decrease the affinity of NifL for ADP significantly, but the mutant proteins exhibit discrete properties. The N419D mutation prevents inhibition of NifA activity by NifL both in vivo and in vitro. In contrast, the G455A and G480A mutations eliminate the redox response, but the mutant proteins retain some sensitivity to the fixed nitrogen status and the ability to interact with the GlnK signal transduction protein. Our data suggest that the absence of the redox switch in the G455A and G480A mutants is a consequence of their inability to override the allosteric effect of 2-oxoglutarate on NifA activity. Overall, these results demonstrate that the binding of adenosine nucleotides to the GHKL domain of NifL plays an important role in counteracting the response of NifA to 2-oxoglutarate, under conditions that are inappropriate for nitrogen fixation.
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PMID:Mutational analysis of the nucleotide-binding domain of the anti-activator NifL. 1570 8

The NifL protein from Azotobacter vinelandii senses both the redox and fixed nitrogen status to regulate nitrogen fixation by controlling the activity of the transcriptional activator NifA. NifL has a domain architecture similar to that of the cytoplasmic histidine protein kinases. It contains two N-terminal PAS domains and a C-terminal transmitter region containing a conserved histidine residue (H domain) and a nucleotide binding GHKL domain corresponding to the catalytic core of the histidine kinases. Despite these similarities, NifL does not exhibit kinase activity and regulates its partner NifA by direct protein-protein interactions rather than phosphorylation. NifL senses the redox status via a FAD co-factor located within the PAS1 domain and responds to the nitrogen status by interaction with the signal transduction protein GlnK, which binds to the GHKL domain. The ability of NifL to inhibit NifA is antagonized by the binding of 2-oxoglutarate to the N-terminal GAF domain of NifA. In this study we have performed site-directed mutagenesis of the H domain of NifL to examine its role in signal transmission. Our results suggest that this domain plays a major role in transmission of signals perceived by the PAS1 and GHKL domains to ensure that NifL achieves the required conformation necessary to inhibit the 2-oxoglutarate-bound form of NifA. Some of the substitutions discriminate the redox and fixed nitrogen sensing functions of NifL implying that the conformational requirements and/or domain interactions necessary for NifA inhibition differ with respect to the signal input.
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PMID:Role of the H domain of the histidine kinase-like protein NifL in signal transmission. 1735 64

The cyanobacterium Synechocystis sp. PCC 6803 harbours 47 histidine kinases (Hiks). Among these are hybrid histidine kinases with one or two response regulator domains as well as numerous Hiks with several sensory domains. One example is the hybrid histidine kinase Slr1759 (Hik14) that has two PAS domains arranged in tandem linked to a predicted GAF domain. Here, we show that a Slr1759 derivative recombinantly expressed in Escherichia coli has a flavin cofactor. Using truncated Slr1759 variants, it is shown that the flavin associates with the first PAS domain. The cofactor reconstitutes the activity of D: -amino acid oxidase apoprotein from pig kidney, indicating that the flavin derivative is FAD. Furthermore, the Slr1759 histidine kinase domain indeed undergoes autophosphorylation in vitro. The phosphorylated product of a recombinant Slr1759 derivative is sensitive to acids, pointing to a histidine residue as the phosphate-accepting group.
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PMID:The hybrid histidine kinase Slr1759 of the cyanobacterium Synechocystis sp. PCC 6803 contains FAD at its PAS domain. 1942 79