Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In Klebsiella pneumoniae NifL antagonizes the action of the transcriptional activator NifA in the presence of molecular oxygen or combined nitrogen. To determine what cofactors might be involved in the oxygen sensing mechanism, we purified and analyzed fusion proteins made between the Escherichia coli maltose binding protein, MalE, and NifL. NifL synthesized and purified under strictly anaerobic conditions did not contain significant amounts of iron or acid-labile sulfur indicating the absence of an oxygen sensing iron-sulfur cluster. However, NifL protein purified in its inhibitory form contained 0.3 +/- 0.01 mol FAD and less than 0.01 mol FMN per mol NifL suggesting the presence of FAD as a cofactor. Characterization of NifL synthesized in the absence of oxygen and combined nitrogen showed that the non-inhibitory form of NifL also contained FAD (0.54 mol FAD per mol NifL). Using fusions between MalE and different portions of NifL we localized the binding site of FAD to the N-terminal domain of NifL. These results and our previous observation that the C-terminal domain of NifL is sufficient to inhibit NifA activity indicate that the N-terminally bound FAD is not directly required for the inhibitory activity of NifL. This observation is supported by the finding that purified apoprotein of NifL was still able to inhibit transcriptional activation by NifA in vitro.
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PMID:NifL of Klebsiella pneumoniae carries an N-terminally bound FAD cofactor, which is not directly required for the inhibitory function of NifL. 943 14

Transcriptional control of the nitrogen fixation (nif) genes in response to oxygen in Azotobacter vinelandii is mediated by nitrogen fixation regulatory protein L (NifL), a regulatory flavoprotein that modulates the activity of the transcriptional activator nitrogen fixation regulatory protein A (NifA). CD spectra of purified NifL indicate that FAD is bound to NifL in an asymmetric environment and the protein is predominantly alpha-helical. The redox potential of NifL is -226 mV at pH 8 as determined by the enzymic reduction of NifL by xanthine oxidase/xanthine in the presence of appropriate mediators. The reduction of NifL by xanthine oxidase prevented NifL from acting as an inhibitor of NifA. In the absence of electron mediators NifL could also be reduced by Escherichia coli flavohaemoprotein (Hmp) with NADH as reductant. Hmp contains a globin-like domain with haem B as prosthetic group and an FAD-containing oxidoreductase module. The carboxyferrohaem form of Hmp was competent to reduce NifL, suggesting that electron donation to NifL originates from the flavin in Hmp rather than by direct electron transfer from the haem. Spinach ferredoxin:NAD(P) oxidoreductase, which adopts a folding similar to the FAD- and NAD-binding domains of Hmp, also reduced NifL with NADH as reductant. Re-oxidation of NifL occurs rapidly in the presence of air, raising the possibility that NifL might sense intracellular oxygen. We propose a physiological redox cycle in which the oxidation of NifL by oxygen and hence the activation of its inhibitory properties occurs rapidly, in contrast with the switch from the active to the reduced form of NifL, which occurs more slowly.
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PMID:Electron donation to the flavoprotein NifL, a redox-sensing transcriptional regulator. 960 Oct 70

In Klebsiella pneumoniae, NifL modulates the activity of the transcriptional activator NifA in response to combined nitrogen or external molecular oxygen. We recently showed that K. pneumoniae NifL is a flavoprotein which apparently senses oxygen through a redox-sensitive, conformational change. In order to study whether the nitrogen signal might be transmitted to NifA through a stable modification of NifL we characterized the redox properties of NifL synthesized in Escherichia coli in the presence of different nitrogen sources. FAD analyses showed that purified NifL carried FAD as cofactor independent of nitrogen and oxygen availability. The redox potential of NifL synthesized in the presence of ammonium was -277+/-5 mV at pH 8.0 and 25 degrees C, as determined by reduction with dithionite or with enzymatic reduction by xanthine oxidase in the presence of methyl viologen as redox mediator. When synthesized under nitrogen-limiting conditions, NifL showed a redox potential of -274+/-6 mV at pH 8.0 and 25 degrees C. Fully reduced NifL fractions, synthesized under either condition listed above, reoxidized rapidly in the presence of molecular oxygen. These results indicate that for NifL synthesized in E. coli, the redox potential of the NifL-bound FAD is not influenced by the nitrogen source. The two NifL fractions differed, however, in that a non-flavin specific absorbance at 420 nm was found only in NifL synthesized in the presence of ammonium.
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PMID:NifL of Klebsiella pneumoniae: redox characterization in relation to the nitrogen source. 1035 Jun 21

This review discusses various mechanisms that regulatory proteins use to control gene expression in response to alterations in redox. The transcription factor SoxR contains stable [2Fe-2S] centers that promote transcription activation when oxidized. FNR contains [4Fe-4S] centers that disassemble under oxidizing conditions, which affects DNA-binding activity. FixL is a histidine sensor kinase that utilizes heme as a cofactor to bind oxygen, which affects its autophosphorylation activity. NifL is a flavoprotein that contains FAD as a redox responsive cofactor. Under oxidizing conditions, NifL binds and inactivates NifA, the transcriptional activator of the nitrogen fixation genes. OxyR is a transcription factor that responds to redox by breaking or forming disulfide bonds that affect its DNA-binding activity. The ability of the histidine sensor kinase ArcB to promote phosphorylation of the response regulator ArcA is affected by multiple factors such as anaerobic metabolites and the redox state of the membrane. The global regulator of anaerobic gene expression in alpha-purple proteobacteria, RegB, appears to directly monitor respiratory activity of cytochrome oxidase. The aerobic repressor of photopigment synthesis, CrtJ, seems to contain a redox responsive cysteine. Finally, oxygen-sensitive rhizobial NifA proteins presumably bind a metal cofactor that senses redox. The functional variability of these regulatory proteins demonstrates that prokaryotes apply many different mechanisms to sense and respond to alterations in redox.
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PMID:Mechanisms for redox control of gene expression. 1054 99

The enzymatic reduction of molecular nitrogen to ammonia requires high amounts of energy, and the presence of oxygen causes the catalyzing nitrogenase complex to be irreversible inactivated. Thus nitrogen-fixing microorganisms tightly control both the synthesis and activity of nitrogenase to avoid the unnecessary consumption of energy. In the free-living diazotrophs Klebsiella pneumoniae and Azotobacter vinelandii, products of the nitrogen fixation nifLA operon regulate transcription of the other nifoperons. NifA activates transcription of nif genes by the alternative form of RNA-polymerase, sigma54-holoenzyme; NifL modulates the activity of the transcriptional activator NifA in response to the presence of combined nitrogen and molecular oxygen. The translationally-coupled synthesis of the two regulatory proteins, in addition to evidence from studies of NifL/NifA complex formation, imply that the inhibition of NifA activity by NifL occurs via direct protein-protein interaction in vivo. The inhibitory function of the negative regulator NifL appears to lie in the C-terminal domain, whereas the N-terminal domain binds FAD as a redox-sensitive cofactor, which is required for signal transduction of the internal oxygen status. Recently it was shown, that NifL acts as a redox-sensitive regulatory protein, which modulates NifA activity in response to the redox-state of its FAD cofactor, and allows NifA activity only in the absence of oxygen. In K. pneumoniae, the primary oxygen sensor appears to be Fnr (fumarate nitrate reduction regulator), which is presumed to transduce the signal of anaerobiosis towards NifL by activating the transcription of gene(s) whose product(s) function to relieve NifL inhibition through reduction of the FAD cofactor. In contrast, the reduction of A. vinelandii-NifL appears to occur unspecifically in response to the availability of reducing equivalents in the cell. Nitrogen status of the cells is transduced towards the NifL/NifA regulatory system by the GlnK protein, a paralogue PII-protein, which appears to interact with the NifL/NifA regulatory system via direct protein-protein interaction. It is not currently known whether GlnK interacts with NifL alone or affects the NifL/NifA-complex; moreover the effects appear to be the opposite in K. pneumoniae and A. vinelandii. In addition to these environmental signals, adenine nucleotides also affect the inhibitory function of NifL; in the presence of ATP or ADP the inhibitory effect on NifA activity in vitro is increased. The NifL proteins from the two organisms differ, however, in that stimulation of K. pneumoniae-NifL occurs only when synthesized under nitrogen excess, and is correlated with the ability to hydrolyze ATP. In general, transduction of environmental signals to the nif regulatory system appears to involve a conformational change of NifL or the NifL/NifA complex. However, experimental data suggest that K. pneumoniae and A. vinelandii employ significantly different species-specific mechanisms of signal transduction.
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PMID:Regulation of nitrogen fixation in Klebsiella pneumoniae and Azotobacter vinelandii: NifL, transducing two environmental signals to the nif transcriptional activator NifA. 1193 53

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

In Klebsiella pneumoniae nitrogen fixation is tightly controlled in response to ammonium and molecular oxygen by the NifL/NifA regulatory system. Under repressing conditions, NifL inhibits the nif-specific transcriptional activator NifA by direct protein-protein interaction, whereas under anaerobic and nitrogen-limited conditions sequestration of reduced NifL to the cytoplasmic membrane impairs inhibition of cytoplasmic NifA by NifL. We report here on a genetic screen to identify amino acids of NifL essential for sequestration to the cytoplasmic membrane under nitrogen-fixing conditions. Overall, 11,500 mutated nifL genes of three independently generated pools were screened for those conferring a Nif(-) phenotype. Based on the respective amino acid changes of nonfunctional derivatives obtained in the screen, and taking structural data into account as well, several point mutations were introduced into nifL by site-directed mutagenesis. The majority of amino acid changes resulting in a significant nif gene inhibition were located in the N-terminal domain (N46D, Q57L, Q64R, N67S, N69S, R80C, and W87G) and the Q-linker (K271E). Further analyses demonstrated that positions N69, R80, and W87 are essential for binding the FAD cofactor, whereas primarily Q64 and N46, but also Q57 and N67, appear to be crucial for direct membrane contact of NifL under oxygen and nitrogen limitation. Based on these findings, we propose that those four amino acids most likely located on the protein surface, as well as the presence of the FAD cofactor, are crucial for the correct overall protein conformation and respective surface charge, allowing NifL sequestration to the cytoplasmic membrane under derepressing conditions.
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PMID:Insights into membrane association of Klebsiella pneumoniae NifL under nitrogen-fixing conditions from mutational analysis. 2105 7

The genes for tetralin (thn) utilization in Sphingomonasmacrogolitabida strain TFA are regulated at the transcriptional level by ThnR, ThnY and ThnA3. ThnR, a LysR-type transcriptional activator activates transcription specifically in response to tetralin, and ThnY is an iron-sulfur flavoprotein that may activate ThnR by protein-protein interaction. ThnA3, a Rieske-type ferredoxin that transfers electrons to the tetralin dioxygenase, prevents transcription of thn genes when the inducer molecule of the pathway is a poor substrate for the dioxygenase. The mechanism by which ThnA3 transduces this signal to the regulatory system is a major question concerning thn gene regulation. Here, we have confirmed the discriminatory function of ThnA3 and the negative role of its reduced form. We have generated ThnY variants with amino acid exchanges in the [2Fe-2S], FAD and NAD(P) H binding domains and their regulatory properties have been analyzed. Two variants, ThnY-C40S and ThnY-N201G,S206P have completely lost the discriminatory function of the regulatory system because they induced thn gene expression with different molecules such us cis-decalin, cyclohexane, trans-decalin, or benzene, which are not real inducers of the pathway. These results support a model in which ThnA3 exerts its negative modulation via the regulator ThnY.
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PMID:The ferredoxin ThnA3 negatively regulates tetralin biodegradation gene expression via ThnY, a ferredoxin reductase that functions as a regulator of the catabolic pathway. 2406 47

D-Glu, an essential component of peptidoglycans, can be utilized as a carbon and nitrogen source by Pseudomonas aeruginosa. DNA microarrays were employed to identify genes involved in D-Glu catabolism. Through gene knockout and growth phenotype analysis, the divergent dguR-dguABC (D-Glu utilization) gene cluster was shown to participate in D-Glu and D-Gln catabolism and regulation. Growth of the dguR and dguA mutants was abolished completely on D-Glu or retarded on D-Gln as the sole source of carbon and/or nitrogen. The dguA gene encoded a FAD-dependent D-amino acid dehydrogenase with d-Glu as its preferred substrate, and its promoter was specifically induced by exogenous D-Glu and D-Gln. The function of DguR as a transcriptional activator of the dguABC operon was demonstrated by promoter activity measurements in vivo and by mobility shift assays with purified His-tagged DguR in vitro. Although the DNA-binding activity of DguR did not require D-Glu, the presence of D-Glu, but not D-Gln, in the binding reaction was found to stabilize a preferred nucleoprotein complex. The presence of a putative DguR operator was revealed by in silica analysis of the dguR-dguA intergenic regions among Pseudomonas spp. and binding of DguR to a highly conserved 19 bp sequence motif was further demonstrated. The dguB gene encodes a putative enamine/imine deaminase of the RidA family, but its role in D-Glu catabolism remains to be determined. Whilst a lesion in dguC encoding a periplasmic solute binding protein only affected growth on D-Glu slightly, expression of the previously characterized AatJMQP transporter for acidic l-amino acid uptake was found inducible by D-Glu and essential for D-Glu utilization. In summary, the findings of this study supported DguA as a new member of the FAD-dependent d-amino acid dehydrogenase family, and DguR as a D-Glu sensor and transcriptional activator of the dguA promoter.
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PMID:Functional characterization of the dguRABC locus for D-Glu and d-Gln utilization in Pseudomonas aeruginosa PAO1. 2508 51


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