A defined amino acid exchange close to the putative nucleotide binding site is responsible for an oxygen-tolerant variant of the Rhizobium meliloti NifA protein.
In Rhizobium meliloti the NifA protein plays a central role in the expression of genes involved in nitrogen fixation. The R. meliloti NifA protein has been found to be oxygen sensitive and therefore acts as a transcriptional activator only under microaerobic conditions. In order to generate oxygen-tolerant variants of the NifA protein a plasmid carrying the R. meliloti nifA gene was mutagenized in vitro with hydroxylamine. About 70 mutated nifA genes were isolated which mediated up to 12-fold increased NifA activity at high oxygen concentrations. A cloning procedure involving the combination of DNA fragments from mutated and wild-type nifA genes allowed mapping of the mutation sites within the central part of the nifA gene. For 17 mutated nifA genes the exact mutation sites were determined by DNA sequence analysis. It was found that all 17 mutated nifA genes carried identical guanosine--adenosine mutations resulting in a methionine--isoleucine exchange (M217I) near the putative nucleotide binding site within the central domain. Secondary structure predictions indicated that the conformation of the putative nucleotide binding site may be altered in the oxygen-tolerant NifA proteins. A model is proposed which assumes that at high oxygen concentrations the loss of activity of the R. meliloti NifA protein is due to a conformational change in the nucleotide binding site that may abolish binding or hydrolysis of the nucleotide. Such a conformational change may be blocked in the oxygen-tolerant NifA protein, thus allowing interaction with the nucleotide at high oxygen concentrations.
Krey
R
R
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Germany.
Pühler
A
A
Klipp
W
W
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009586:Nitrogen Fixation / Q000235:genetics*; D009711:Nucleotides / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D015183:Restriction Mapping; D016962:Sinorhizobium meliloti / Q000235:genetics*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006898:Hydroxylamines; C061338:NifA protein, Bacteria; D009711:Nucleotides; D014157:Transcription Factors; D019811:Hydroxylamine; D000255:Adenosine Triphosphate; D010100:Oxygen