Altered kinetic properties of tyrosine-183 to cysteine mutation in glutamine synthetase of anabaena variabilis strain SA1 is responsible for excretion of ammonium ion produced by nitrogenase.
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 NH(4)(+)-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 K(m) values at pH 7.5: glutamate, 1.7 m M; NH(4)(+), 0.015 m M; ATP, 0.13 m M. The apparent K(m) for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 m M; NH(4)(+), 12.4 m M; ATP, 0.17 m M). 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 K(m) 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.
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
Franklyn G
FG
Healy
Charles
C
Latorre
Stephan L
SL
Albrecht
Pallavolu M
PM
Reddy
Keelnatham T
KT
Shanmugam
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017033:Anabaena / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D009591:Nitrogenase / Q000378:metabolism*; D017354:Point Mutation; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D016415:Sequence Alignment; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D000644:Quaternary Ammonium Compounds; D014443:Tyrosine; D008345:Manganese; D009591:Nitrogenase; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium; D003545:Cysteine