Mutational analysis of Asp51 of Anabaena azollae glutamine synthetase.

Abstract

The role of Asp51 in the catalytic activity of glutamine synthetase from the cyanobacterium Anabaena azollae has been analyzed. Five mutant enzymes, D51S, D51A, D51E, D51N and D51R, were constructed by sitedirected mutagenesis and characterized. Asp51 appears to participate in the binding of ammonium ion, as affinity for this substrate was affected in all cases, although it varied according to the charge and/or size of the amino-acid residue, decreasing in the order Glu . Asn . Ser . Ala. The replacement of Asp51 by Glu (D51E) conferred besides a high resistance to the herbicides l-methionine-dl-sulfoximine and phosphinothricin, as a result of a decreased phosphorylation ability.

Metabolic engineering of ammonium release for nitrogen-fixing multispecies microbial cell-factories

“…For site-directed mutagenesis of A. vinelandii GS encoded by the glnA gene, sequence alignments of GSs were carried out to identify a candidate amino acid in A. vinelandii GS that might display the critical function of D50 or D51 in ammonium binding and deprotonation as in E. coli (Liaw et al, 1995; or Anabaena azollae (Crespo et al, 1999) GSs, respectively. Thus the candidate D49 was identified for A. vinelandii GS.…”

“…Previous work in E. coli and Salmonella typhimurium , and Anabaena azollae (Crespo et al, 1999) GS, showed that residues D50 or D51, respectively, are involved in ammonium binding and deprotonation at the active site of GS in these bacteria. Thus, by multiple sequence alignments we identified D49 in A. vinelandii GS as a candidate amino acid for a site-directed mutagenesis approach and obtained the A. vinelandii single mutant strain D49S, AV6 and double mutant strain ΔnifL-D49S, AV7 (Fig.…”