Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense
| Autor: | Jeremy Thornton, Juliana Inaba, Rose A. Monteiro, Luciano F. Huergo, Emanuel Maltempi de Souza, Giseli Klassen, Fábio O. Pedrosa, Mike Merrick |
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| Rok vydání: | 2015 |
| Předmět: |
Protein Conformation
PII Nitrogen Regulatory Proteins DNA Mutational Analysis Mutant Gene Expression Mutagenesis (molecular biology technique) Azospirillum brasilense medicine.disease_cause Microbiology Adenosine Triphosphate Bacterial Proteins Nitrogenase medicine Escherichia coli Gene biology Wild type biology.organism_classification A-site Amino Acid Substitution Biochemistry Mutation Protein Binding Transcription Factors |
| Zdroj: | Microbiological Research. 171:65-72 |
| ISSN: | 0944-5013 |
| DOI: | 10.1016/j.micres.2014.12.005 |
| Popis: | PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ. In this paper, glnB was mutagenised in order to identify amino acid residues involved in GlnB signaling. Two variants were obtained by random mutagenesis, GlnBL13P and GlnBV100A and a site directed mutant, GlnBY51F, was obtained. Their ability to complement nitrogenase activity of glnB mutant strains of A. brasilense were determined. The variant proteins were also overexpressed in Escherichia coli, purified and characterized biochemically. None of the GlnB variant forms was able to restore nitrogenase activity in glnB mutant strains of A. brasilense LFH3 and 7628. The purified GlnBY51F and GlnBL13P proteins could not be uridylylated by GlnD, whereas GlnBV100A was uridylylated but at only 20% of the rate for wild type GlnB. Biochemical and computational analyses suggest that residue Leu13, located in the α helix 1 of GlnB, is important to maintain GlnB trimeric structure and function. The substitution V100A led to a lower affinity for ATP binding. Together the results suggest that NifA activation requires uridylylated GlnB bound to ATP. |
| Databáze: | OpenAIRE |
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