Mutational analysis of Asp51 of Anabaena azollae glutamine synthetase. D51E mutation confers resistance to the active site inhibitors L-methionine-DL-sulfoximine and phosphinothricin.
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 site-directed 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.
Crespo
J L
JL
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Sevilla, Spain.
Guerrero
M G
MG
Florencio
F J
FJ
D000595:Amino Acid Sequence; D000613:Aminobutyrates / Q000494:pharmacology; D017033:Anabaena / Q000201:enzymology* / Q000235:genetics*; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D006540:Herbicides / Q000494:pharmacology; D007700:Kinetics; D008717:Methionine Sulfoximine / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid
D000613:Aminobutyrates; D017931:DNA Primers; D004791:Enzyme Inhibitors; D006540:Herbicides; D008717:Methionine Sulfoximine; D001224:Aspartic Acid; C003121:phosphinothricin; D005974:Glutamate-Ammonia Ligase