Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL

Austin, Sara; Buck, Martin; Cannon, Wendy; Eydmann, Trevor; Dixon, Ray

doi:10.1128/jb.176.12.3460-3465.1994

Cited by 85 publications

(107 citation statements)

References 26 publications

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“…The ␤-galactosidase assays were carried out as described (29,35) Protein Expression and Purification. Plasmids pTJ45, pDB737, and pIM15 were used for overexpression of N his6 NifL, NifA, and NifA-E356K (28)(29)(30). For overexpression of N his6 NifL-R306C, an NdeI-BamHI fragment encoding the corresponding mutation was cloned into plasmid pET28, resulting in plasmid pIM22.…”

Section: Methodsmentioning

confidence: 99%

“…Although NifL contains an H-box motif (Fig. 1), the conserved histidine residue is not required for regulation of NifA activity (26) and neither autophosphorylation of NifL nor phosphotransfer to NifA has been detected (27,28). These observations, together with the fact that NifA is not a member of the response-regulator family, suggest that NifL is not a bona fide HPK.…”

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confidence: 96%

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A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii

Martínez‐Argudo

Little

Dixon

2004

Proc. Natl. Acad. Sci. U.S.A.

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NifL is an antiactivator that tightly regulates transcription of genes required for nitrogen fixation in Azotobacter vinelandii by controlling the activity of its partner protein NifA, a member of the family of 54 -dependent transcriptional activators. Although the C-terminal region of A. vinelandii NifL shows homology to the transmitter domains of histidine protein kinases, signal transduction between NifL and NifA is conveyed by means of proteinprotein interactions rather than by phosphorylation. Binding of the ligand 2-oxoglutarate to NifA plays a crucial role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. We have used a suppressor screen to identify a critical arginine residue (R306) in NifL that is required to release NifA from inhibition under appropriate environmental conditions. Amino acid substitutions at position 306 result in constitutive inhibition of NifA activity by NifL, thus preventing nitrogen fixation. Biochemical studies with one of the mutant proteins demonstrate that the substitution alters the conformation of NifL significantly and prevents the response of NifA to 2-oxoglutarate. We propose that arginine 306 is critical for the propagation of signals perceived by A. vinelandii NifL in response to the redox and fixed-nitrogen status and is required for a conformational switch that inactivates the inhibitory function of NifL under conditions appropriate for nitrogen fixation.2-oxoglutarate ͉ signal transduction ͉ antiactivator ͉ redox control ͉ nitrogen regulation S tructural rearrangements of sensor proteins in response to environmental cues provide a fundamental mechanism for signal propagation within cells. However, although conformational changes have been well characterized in isolated signaling domains, mechanisms for signal transmission by means of interdomain interactions are frequently less well understood. For example, structural studies have identified ligand-induced conformational changes in the sensor domains of histidine protein kinases (HPKs), but it is not known how these changes are communicated to the kinase domain to control phosphoryl transfer.The Azotobacter vinelandii NifL regulatory protein is a histidine kinase-like protein that controls the expression of the genes required for nitrogen fixation in response to the redox, nitrogen, and carbon status. NifL is an antiactivator that tightly regulates the activity of its partner protein NifA, a member of the family of 54 -transcriptional activators (1, 2), by means of the formation of an inhibitory complex (3-5). The domain architecture of NifL is similar to that of some HPKs, with an N-terminal Per-ArntSim (PAS) domain (6, 7) containing a flavin adenine dinucleotide (FAD) cofactor that senses the redox status (8, 9) and a C-terminal domain containing conserved residues corresponding to the N, G1, F, and G2 boxes that constitute the ATPbinding domain of the GHKL superfamily of ATPases (10-14) (Fig. 1). Unlike the HPKs, the GHKL domain of NifL does not exhibit ATP hydrolysis or transphos...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 96%

A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii

Martínez‐Argudo

Little

Dixon

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Plasmids for Protein Overexpression-Plasmid pTJ42 expressing native A. vinelandii NifA with a C-terminal hexahistidine tag (NifA 6-His ) was prepared from plasmid pDB737 (28). An EcoRI-BamHI fragment from pDB737 containing the 3Ј end of NifA was cloned into the vector pTE103 to give plasmid pTJ39 containing a single BglII site within the EcoRI-BamHI region.…”

Section: Methodsmentioning

confidence: 99%

“…4A). Although the kinase-like domain of NifL binds adenosine nucleotides (2), no autokinase or phosphotransferase activities have been detected in vitro (28). Nevertheless, the similarity between NifL and the histidine protein kinase family is of interest, in light of the recent demonstration that the Cterminal kinase domain of NtrB (NRII) interacts with enteric PII (35,38,39).…”

Section: Mutant Forms Of Av Glnk Defective In Regulating Nifl-nifamentioning

confidence: 99%

Direct Interaction of the NifL Regulatory Protein with the GlnK Signal Transducer Enables the Azotobacter vinelandiiNifL-NifA Regulatory System to Respond to Conditions Replete for Nitrogen

Little

Colombo

Leech

et al. 2002

Journal of Biological Chemistry

107

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“…Some predicted homologs of histidine kinases may not be kinases or may not phosphorylate a histidine residue (32). For example, Azotobacter vinelandii NifL contains five conserved blocks of amino acids characteristic of histidine sensor kinases (39) but has not been observed to autophosphorylate (40,41), and DivL (42), pyruvate dehydrogenase kinase (43), and plant phytochromes (44) phosphorylate a tyrosine or serine rather than a histidine.…”

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confidence: 99%

A Two-component System Mediates Developmental Regulation of Biosynthesis of a Heterocyst Polysaccharide

Zhou

Wölk

2003

Journal of Biological Chemistry

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Some cyanobacteria couple oxygenic photosynthesis in vegetative cells with O 2 -sensitive N 2 fixation in differentiated cells called heterocysts. Heterocyst differentiation involves extensive biochemical and structural changes that collectively permit heterocysts to assimilate N 2 aerobically and supply the products of N 2 fixation to vegetative cells. HepK and DevR are required for the development of functional heterocysts in Anabaena and Nostoc, respectively. We show that HepK is an autokinase and that Anabaena DevR A is its cognate response regulator, together comprising part or all of a two-component system that mediates developmental regulation of biosynthesis of a heterocyst envelope polysaccharide. Recombinant N-hexahistidine-tagged

show abstract

Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL

Cited by 85 publications

References 26 publications

A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii

A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii

Direct Interaction of the NifL Regulatory Protein with the GlnK Signal Transducer Enables the Azotobacter vinelandiiNifL-NifA Regulatory System to Respond to Conditions Replete for Nitrogen

A Two-component System Mediates Developmental Regulation of Biosynthesis of a Heterocyst Polysaccharide

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