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(A) Summary of regions protected from trypsin digestion in the NIFL-NIFA complex formed in the presence of MgADP. The hatched areas signify protection from protease treatment. (B) Model of the interaction of NIFL with NIFA in the presence of MgADP.
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The enhancer binding protein NIFA and the sensor protein NIFL fromAzotobacter vinelandii comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the
two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity
of NIFL to...
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In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition...
Citations
... On the other hand, the C-terminal domain of NifL has been found to bind ADP (S€ oderb€ ack et al., 1998) and it was the C-terminal domain of NifL that interacted with the N-terminal domain of NifA (Money, Jones, Dixon, & Austin, 1999). A complex of purified NifA and NifL formed in presence of MgADP, when subjected to limited proteolysis, revealed protection of the N-terminal region of NifA close to the Q-linker (Money, Barrett, Dixon, & Austin, 2001). Interestingly, NifL devoid of the first 146 amino acids could counteract NifA activity in vitro in response to ADP and also in response to fixed nitrogen, but not in response to oxygen. ...
Azotobacters have been used as biofertilizer since more than a century. Azotobacters fix nitrogen aerobically, elaborate plant hormones, solubilize phosphates and also suppress phytopathogens or reduce their deleterious effect. Application of wild type Azotobacters results in better yield of cereals like corn, wheat, oat, barley, rice, pearl millet and sorghum, of oil seeds like mustard and sunflower, of vegetable crops like tomato, eggplant, carrot, chillies, onion, potato, beans and sugar beet, of fruits like mango and sugar cane, of fiber crops like jute and cotton and of tree like oak. In addition to the structural genes of the enzyme nitrogenase and of other accessory proteins, A. vinelandii chromosomes contain the regulatory genes nifL and nifA. NifA must bind upstream of the promoters of all nif operons for enabling their expression. NifL on activation by oxygen or ammonium, interacts with NifA and neutralizes it. Nitrogen fixation has been enhanced by deletion of nifL and by bringing nifA under the control of a constitutive promoter, resulting in a strain that continues to fix nitrogen in presence of urea fertilizer. Additional copies of nifH (the gene for the Fe-protein of nitrogenase) have been introduced into A. vinelandii, thereby augmenting nitrogen fixation. The urease gene complex ureABC has been deleted, the ammonia transport gene amtB has been disrupted and the expression of the glutamine synthase gene has been regulated to enhance urea and ammonia excretion. Gluconic acid has been produced by introducing the glucose dehydrogenase gene, resulting in enhanced solubilization of phosphate.
... We have previously observed changes in the pattern of chymotrypsin proteolysis when comparing oxidized and reduced forms of NifL (1–284), a protein fragment containing both the PAS1 and PAS2 domains (Slavny et al., 2010 ). The major chymotrypsin sensitive sites are primarily located in PAS2 (Money et al., 2001) and the pattern of digestion reflects redox-dependent conformational changes dependent upon the redox state of the FAD cofactor in PAS1 (Slavny et al., 2010). Different mutant classes can therefore be discriminated and subsequently compared with the equivalent wild-type signalling state. ...
The Per-ARNT-Sim (PAS) domain is a conserved α/β fold present within a plethora of signalling proteins from all kingdoms of life. PAS domains are often dimeric and act as versatile sensory and interaction modules to propagate environmental signals to effector domains. The NifL regulatory protein from Azotobacter vinelandii senses the oxygen status of the cell via an FAD cofactor accommodated within the first of two amino-terminal tandem PAS domains, termed PAS1 and PAS2. The redox signal perceived at PAS1 is relayed to PAS2 resulting in conformational reorganization of NifL and consequent inhibition of NifA activity. We have identified mutations in the cofactor-binding cavity of PAS1 that prevent 'release' of the inhibitory signal upon oxidation of FAD. Substitutions of conserved β-sheet residues on the distal surface of the FAD-binding cavity trap PAS1 in the inhibitory signalling state, irrespective of the redox state of the FAD group. In contrast, substitutions within the flanking A'α-helix that comprises part of the dimerization interface of PAS1 prevent transmission of the inhibitory signal. Taken together, these results suggest an inter-subunit pathway for redox signal transmission from PAS1 that propagates from core to the surface in a conformation-dependent manner requiring a flexible dimer interface.
... This finding strongly indicates that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae (55). In Azotobacter vinelandii, another diazotrophic bacterium regulating its nif gene expression by a nifLA operon, NifA activity is also regulated in response to ammonium and molecular oxygen by direct protein-protein interaction by its negative regulator NifL (4,23,39,40,47). However, in contrast to K. pneumoniae a change in the cellular localization of NifL has not been observed, and the electrons for FAD reduction are presumably derived from reduction equivalents present in the cytoplasm during anaerobiosis (7,30,34,35). ...
... Changing position 271 in the Q-linker did not significantly affect the ability to associate with liposomes; however, the strong decrease in nif induction indicates that the Q-linker of K. pneumoniae plays an important role for the overall NifL conformation crucial for interacting with NifA. In accordance with this, Austin and coworkers proposed for A. vinelandii NifL that the Q-linker is probably involved in conformational changes, resulting in a conformation that interacts with its target NifA under repressing conditions (39). Thus, it is attractive to speculate that changing position 271 in the Q-linker increases stability of inhibitory NifL/NifA complexes in K. pneumoniae even under nitrogen-fixing conditions leading to a reduced nif gene induction. ...
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.
... A fragment at 19 kDa in the holo-form is the other major difference, although this was hardly observed in the apo-form. Regarding the effect of AMP-PNP on the proteolysis of VnfA, a similar effect of the nucleotide binding was reported in a study of the limited trypsin digestion with NifA + MgADP, in which binding MgADP to the central AAA+ domain is ascribed to the trigger of a conformational change of NifA to avoid further proteolysis [34,35]. By analogy with the study on NifA, the observed transformation of VnfA to a more resistant form to proteolysis is ascribed to a conformational change induced by binding AMP-PNP, presumably at the central domain of VnfA. ...
... Although the signal at g = 2.03 still shifted to 2.02 and a fully identical spectrum to that observed in the whole cell measurement has not been reproduced under the present reconstitution conditions, the partial recovery of the rhombicity implies that VnfA can bind a nucleotide, and the whole cell EPR spectrum might reflect VnfA of the nucleotide binding form. It has been reported that binding of ATP or ADP to NifA of A. vinelandii leads to rearrangement of interaction between the GAF and AAA+ domains (and thereby a conformational change in the protein), which are considered to couple with transmission processes of the sensing events [34]. Considering the functional and structural analogies to NifA, it is presumably rational to expect that VnfA also causes a conformational change in a similar manner to NifA; the binding of the ATP analog induces the rearrangement of the GAF and possible AAA+ (the central domain) domains in VnfA. ...
... Limited protease digestion assays were carried out in a mixture containing 50 mm Tris-acetate (pH 8.0), 100 lm VnfA contains an iron-sulfur cluster potassium acetate, 8 mm magnesium acetate and 1 mm dithiothreitol at 20°C in the presence or absence of 3 mm AMP-PNP according as described previously [34]. After preincubation of apo-or reconstituted VnfA (18 lg) with or without AMP-PNP for 2 min, the reactions were started by the addition of Tripsin (0.1 lg). ...
Transcriptional activator VnfA is required for the expression of a second nitrogenase system encoded in the vnfH and vnfDGK operons in Azotobacter vinelandii . In the present study, we have purified full‐length VnfA produced in E. coli as recombinant proteins ( Strep ‐tag attached and tag‐less proteins), enabling detailed characterization of VnfA for the first time. The EPR spectra of whole cells producing tag‐less VnfA (VnfA) show distinctive signals assignable to a 3Fe‐4S cluster in the oxidized form ([Fe 3 S 4 ] ⁺ ). Although aerobically purified VnfA shows no vestiges of any Fe‐S clusters, enzymatic reconstitution under anaerobic conditions reproduced [Fe 3 S 4 ] ⁺ dominantly in the protein. Additional spectroscopic evidence of [Fe 3 S 4 ] ⁺ in vitro is provided by anaerobically purified Strep ‐tag attached VnfA. Thus, spectroscopic studies both in vivo and in vitro indicate the involvement of [Fe 3 S 4 ] ⁺ as a prosthetic group in VnfA. Molecular mass analyses reveal that VnfA is a tetramer both in the presence and absence of the Fe‐S cluster. Quantitative data of iron and acid‐labile sulfur in reconstituted VnfA are fitted with four 3Fe‐4S clusters per a tetramer, suggesting that one subunit bears one cluster. In vivo β‐gal assays reveal that the Fe‐S cluster which is presumably anchored in the GAF domain by the N‐terminal cysteine residues is essential for VnfA to exert its transcription activity on the target nitrogenase genes. Unlike the NifAL system of A. vinelandii , O 2 shows no effect on the transcriptional activity of VnfA but reactive oxygen species is reactive to cause disassembly of the Fe‐S cluster and turns active VnfA inactive.
Structured digital abstract
MINT‐7311946 : VnfA (uniprotkb: C1DI41 ) and VnfA (uniprotkb: C1DI41 ) bind ( MI:0407 ) by molecular sieving ( MI:0071 )
MINT‐7311931 : VnfA (uniprotkb: C1DI41 ) and VnfA (uniprotkb: C1DI41 ) bind ( MI:0407 ) by blue native page ( MI:0276 )
... Approximate molecular weights determined by calibrations with markers are listed to the left of (A) and (D). Open arrowheads indicate putative proteolytic fragments arising from digestion at the chymotrypsin-sensitive sites F218 and F222 identified previously (Money et al., 2001). NifL(1-284) adopts a similar conformation to that of NifL (1-284)-I153A, while differing in conformation to NifL (1-284)-V166M. ...
Per-Arnt-Sim (PAS) domains play a critical role in signal transduction in multidomain proteins by sensing diverse environmental signals and regulating the activity of output domains. Multiple PAS domains are often found within a single protein. The NifL regulatory protein from Azotobacter vinelandii contains tandem PAS domains, the most N-terminal of which, PAS1, contains a FAD cofactor and is responsible for redox sensing, whereas the second PAS domain, PAS2, has no apparent cofactor and its function is unknown. Amino acid substitutions in PAS2 were identified that either lock NifL in a form that constitutively inhibits NifA or that fail to respond to the redox status, suggesting that PAS2 plays a pivotal role in transducing the redox signal from PAS1 to the C-terminal output domains. The isolated PAS2 domain is a homodimer in solution and the subunits are in rapid exchange. PAS2 dimerization is maintained in the redox signal transduction mutants, but is inhibited by substitutions in PAS2 that lock NifL in the inhibitory conformer. Our results support a model for signal transduction in NifL, whereby redox-dependent conformational changes in PAS1 are relayed to the C-terminal domains via changes in the quaternary structure of the PAS2 domain.
... Similar to K. pneumoniae, the N 2 fixation of A. vinelandii is under two types of regulation: global and nif specific (19,26,28,43). The nif-specific regulation is by the nifLA operon and is very complex (18,32,45,46). The activity of nifA is decreased following interaction with the nifL gene product. ...
Nitrogen fixation in Azotobacter vinelandii is regulated by the nifLA operon. NifA activates the transcription of nif genes, while NifL antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen levels.
However, transcriptional regulation of the nifLA operon of A. vinelandii itself is not fully understood. Using the S1 nuclease assay, we mapped the transcription start site of the nifLA operon, showing it to be similar to the σ54-dependent promoters. We also identified a positive cis-acting regulatory element (+134 to +790) of the nifLA operon within the coding region of the nifL gene of A. vinelandii. Deletion of this element results in complete loss of promoter activity. Several protein factors bind to this region, and
the specific binding sites have been mapped by DNase I foot printing. Two of these sites, namely dR1 (+134 to +204) and dR2
(+745 to +765), are involved in regulating the nifLA promoter activity. The absence of NtrC-like binding sites in the upstream region of the nifLA operon in A. vinelandii makes the identification of these downstream elements a highly significant finding. The interaction of the promoter with
the proteins binding to the dR2 region spanning +745 to +765 appears to be dependent on the face of the helix as introduction
of 4 bases just before this region completely disrupts promoter activity. Thus, the positive regulatory element present within
the BglII-BglII fragment may play, in part; an important role in nifLA regulation in A. vinelandii.
... BH72, NifA transcriptional activity is regulated by a second regulatory protein, NifL, which inhibits NifA in response to external molecular oxygen and ammonium [5][6][7][8]. This inhibition of NifA activity by NifL apparently occurs via direct protein-protein interaction, which is implied by evidence from immunological studies in K. pneumoniae [9], and is consistent with recent studies for A. vinelandii using the yeast two-hybrid system and in vitro analysis of complex formation between NifL and NifA [10][11][12][13][14]. ...
... Rechromatography further showed that up to 90% of the isolated complexes bound again to amylose resin, indicating that NifL-NifA complexes formed in vivo are stable and do not rapidly dissociate upon storage at 4°C. These findings indicate that stable complexes between K. pneumoniae NifA and NifL are formed exclusively in vivo under physiological conditions, which is in contrast to A. vinelandii [10,11]. Alternatively, for K. pneumoniae bridging proteins might be necessary for complex formation between NifL and NifA, which are missing in the in vitro analysis. ...
In Klebsiella pneumoniae, the nif specific transcriptional activator NifA is inhibited by NifL in response to molecular oxygen and ammonium. Here, we demonstrate complex formation between NifL and NifA (approximately 1 : 1 ratio), when synthesized in the presence of oxygen and/or ammonium. Under simultaneous oxygen- and nitrogen-limitation, significant but fewer NifL-NifA complexes (approximately 1%) were formed in the cytoplasm as a majority of NifL was sequestered to the cytoplasmic membrane. These findings indicate that inhibition of NifA in the presence of oxygen and/or ammonium occurs via direct NifL interaction and formation of those inhibitory NifL-NifA complexes appears to be directly and exclusively dependent on the localization of NifL in the cytoplasm. We further observed evidence that the nitrogen sensory protein GlnK forms a trimeric complex with NifL and NifA under nitrogen limitation. Binding of GlnK to NifL-NifA was specific; however the amount of GlnK within these complexes was small. Finally, two lines of evidence were obtained that under anaerobic conditions but in the presence of ammonium additional NtrC-independent GlnK synthesis inhibited the formation of stable inhibitory NifL-NifA complexes. Thus, we propose that the NifL-NifA-GlnK complex reflects a transitional structure and hypothesize that under nitrogen-limitation, GlnK interacts with the inhibitory NifL-NifA complex, resulting in its dissociation.
... kDa polypeptides (which represent the central plus the carboxyl-terminal domain and the isolated central domain, respectively) and a 20-kDa polypeptide designated A6, which is a central domain subdomain fragment (Fig. 4A) (26). The amino termini of these polypeptides result from cleavage at Arg-202, located within the linker between the GAF and central domains. ...
... The amino termini of these polypeptides result from cleavage at Arg-202, located within the linker between the GAF and central domains. We have also detected cleavages within the GAF domain itself at Arg-8, Arg-70, and Arg-165 (26). We performed similar experiments to determine whether 2-oxoglutarate influences the pattern of proteolytic digestion of the GAF domain. ...
... We have shown previously that when the NifL-NifA complex is formed, NifA is protected from proteolysis at Arg-202 and within the carboxyl-terminal region of the GAF domain, suggesting that NifL may restrict access to these cleavage sites or induce a conformational change (26). Since the binding of 2-oxoglutarate to NifA prevents inhibition by NifL and potentially inhibits the interaction between the two proteins, we re-examined the influence of NifL on proteolysis of NifA in the presence of 2-oxoglutarate. ...
The expression of genes required for the synthesis of molybdenum nitrogenase in Azotobacter vinelandii is controlled by the NifL-NifA transcriptional regulatory complex in response to nitrogen, carbon, and redox status. Activation of nif gene expression by the transcriptional activator NifA is inhibited by direct protein-protein interaction with NifL under conditions unfavorable for nitrogen fixation. We have recently shown that the NifL-NifA system responds directly to physiological concentrations of 2-oxoglutarate, resulting in relief of NifA activity from inhibition by NifL under conditions when fixed nitrogen is limiting. The inhibitory activity of NifL is restored under conditions of excess combined nitrogen through the binding of the signal transduction protein Av GlnK to the carboxyl-terminal domain of NifL. The amino-terminal domain of NifA comprises a GAF domain implicated in the regulatory response to NifL. A truncated form of NifA lacking this domain is not responsive to 2-oxoglutarate in the presence of NifL, suggesting that the GAF domain is required for the response to this ligand. Using isothermal titration calorimetry, we demonstrate stoichiometric binding of 2-oxoglutarate to both the isolated GAF domain and the full-length A. vinelandii NifA protein with a dissociation constant of approximately 60 microm. Limited proteolysis experiments indicate that the binding of 2-oxoglutarate increases the susceptibility of the GAF domain to trypsin digestion and also prevents NifL from protecting these cleavage sites. However, protection by NifL is restored when the non-modified (non-uridylylated) form of Av GlnK is also present. Our results suggest that the binding of 2-oxoglutarate to the GAF domain of NifA may induce a conformational change that prevents inhibition by NifL under conditions when fixed nitrogen is limiting.
... The locations of the hydrophilic interdomain linker (Q-linker), the PAS domain, and the apparent ATP binding site are indicated. et al., 1999; Lei et al., 1999; Money et al., 2001). Thus, it appears that signal transduction apparently occurs via protein-protein interaction. ...
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.
... The nif-specific transcriptional activator, NifA, activates transcription by 54 ( N )-RNA polymerase holoenzyme at nif promoters under conditions appropriate for nitrogen fixation, and the regulatory protein NifL controls the transcriptional activation functions of NifA in response to environmental cues (1). The NifL protein utilizes discrete mechanisms to perceive these signals (2), leading to the formation of a protein-protein complex that inhibits NifA activity (3,4). The binding of adenosine nucleotides to NifL plays a key role in transducing environmental signals to form the inhibitory protein complex (3,5). ...
... Although A. vinelandii NifL does not apparently hydrolyze ATP and neither kinase nor autophosphorylation activities have been detected, the Cterminal region of NifL does show significant homology to the transmitter region of the histidine protein kinases and in particular contains the four conserved regions designated N, G 1 , F, and G 2 , which are implicated in nucleotide binding (36,37). Because adenosine nucleotides bind to the kinase-like domain to stimulate the NifL-NifA interaction, it is possible that this domain of NifL represents either an ancient precursor of the histidine protein kinases or has evolved from the "classical" kinases with loss of catalytic function (nucleoside triphosphate hydrolysis), so that nucleotide binding is utilized to drive conformational changes that promote interaction with NifA (3,4). The homology between the kinase-like domains of NifL and NtrB (NRII) is of particular interest in view of the recent finding that this domain of NtrB (residues 190 -339) interacts with Ec PII (35,38). ...
The Azotobacter vinelandii NifL-NifA regulatory system integrates metabolic signals for redox, energy, and nitrogen status to fine tune regulation of
the synthesis of molybdenum nitrogenase. The NifL protein utilizes discrete mechanisms to perceive these signals leading to
the formation of a protein-protein complex, which inhibits NifA activity. Whereas redox signaling is mediated via a flavin-containing
PAS domain in the N-terminal region of NifL, the nitrogen status is sensed via interaction with PII-like signal transduction
proteins and small molecular weight effectors. The nonuridylylated form of the PII-like protein encoded by A. vinelandii glnK (Av GlnK) stimulates NifL to inhibit transcriptional activation by NifA in vitro. Here we demonstrate that the nonmodified form of Av GlnK directly interacts with A. vinelandii NifL and that this interaction is dependent on Mg2+, ATP, and 2-oxoglutarate. Differences were observed in the regulation of the Av GlnK-NifL interaction by 2-oxoglutarate compared
with the role of this effector in modulating the interaction of enteric PII-like proteins with their receptors. We also report
that the interaction between Av GlnK and NifL is abolished by site-directed substitution of a single amino acid in the T-loop
region of Av GlnK and that uridylylation of the conserved tyrosine residue in the T-loop inhibits the interaction. No association
was detected between Av GlnK and the N-terminal region of NifL and our results demonstrate that Av GlnK directly interacts
with the C-terminal histidine protein kinase-like domain.
