Two residues in the T-loop of GlnK determine NifL-dependent nitrogen control of nif gene expression.
X-ray crystallographic analysis of the Escherichia coli P(II) protein paralogues GlnB and GlnK has shown that they share a superimposable structural core but can differ in conformation of the T-loop, a region of the protein (residues 37-54) that has been shown to be important for interaction with other proteins. In Klebsiella pneumoniae GlnK has been shown to have a clearly defined function in regulating NifL-mediated inhibition of NifA activity in response to the nitrogen status, and GlnB, when expressed from the chromosome, does not substitute for GlnK. Because the T-loops of K. pneumoniae and E. coli GlnB and GlnK differ at just three residues, 43, 52, and 54, we have used a previously constructed heterologous system, in which K. pneumoniae nifLA is expressed in E. coli, to investigate the importance of GlnK residues 43, 52, and 54 for regulation of the NifLA interaction. By site-directed mutagenesis of glnB we have shown that residue 54 is the single most important amino acid in the T-loop in the context of the regulation of NifA activity. Furthermore, a combination of just two changes, in residues 54 and 43, allows GlnB to function as GlnK and completely relieve NifL inhibition of NifA activity.
Arcondeguy
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Departments of Molecular Microbiology and Biological Chemistry, John Innes Centre, Norwich NR4 7UH, United Kingdom.
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Merrick
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D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005838:Genotype; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009586:Nitrogen Fixation / Q000235:genetics*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; C113488:GlnK protein, Azorhizobium caulinodans; D011994:Recombinant Proteins; C084296:nifL protein, Bacteria