Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/29/2002
Publication Date: 5/14/2003
Citation: HEALY, F.G., LATORRE, C., ALBRECHT, S.L., REDDY, P.M., SHANMUGAM, K.T. 2003. ALTERED KINETIC PROPERTIES OF TYROSINE-183 TO CYSTEINE MUTATION IN GLUTAMINE SYNTHETASE OF ANABAENA VERIABILIS STRAIN SA1 IS RESPONSIBLE FOR THE EXCRETION OF AMMONIUM ION PRODUCED BY NITROGENASE. CURRENT MICROBIOLOGY. 46(2003):423-431.
Interpretive Summary: Some bacteria can convert atmospheric nitrogen (nitrogen fixation) into a form that can be used by crops. This conversion requires energy, but some nitrogen-fixing bacteria are photosynthetic, like higher plants, and can use light energy to make the conversion of nitrogen gas to ammonia. In future the ability to use these organisms in agriculture may reduce fertilizer costs for farmers. It is important to understand the biochemistry of these organisms to fully utilize their potential. Several mutants of a strain of the photosynthetic bacteria Anabaena variabilis were produced and one of the mutants was defective in its ability of convert nitrogen to ammonia. The defect was found in an enzyme (glutamine synthetase) that is involved in nitrogen assimilation. This defect caused ammonia to be released from the bacteria, and it was demonstrated that the liberated ammonia could be used to fertilize rice plants. To understand the biochemical basis for the ammonia excretion, the enzyme from both the parent and the mutant strain was purified. The enzymes from the different strains displayed different kinetic properties and different levels of inhibition by selected amino acids. The genetic material encoding the enzyme (glutamine synthetase) from each strain was isolated, amplified and compared by nucleotide sequencing. The sequences from both strains were identical except for a single nucleotide substitution and demonstrate the importance of structural changes on enzyme activity.
Technical Abstract: A L-methionine-D,L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N_2 reduction. In order to determine the biochemical basis for the NH4^+-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required Mn^+2 for optimum activity, while SA0-GS was Mg^+2 dependent. SA0-GS had the following apparent Km values at pH 7.5: glutamate, 1.7 mM; NH_46^+, 0.015 mM; ATP, 0.13 mM. The apparent Km for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 mM; NH_4^+, 12.4 mM; ATP, 0.17 mM). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH_4^+-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the Km values for NH_4^+ and glutamate, and thus limited the assimilation of NH_4^+ generated by N_2 reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH_4^+ binding and metal ion coordination.