Isolation and characterization of a novel glutamine synthetase from Rhizobium meliloti.
Two glutamine synthetases, GSI and GSII, are found in most rhizobia. However, WSU650, a Rhizobium meliloti glnA glnII mutant that lacks both enzymes, can grow without a glutamine supplement in minimal medium that contains both ammonium and glutamate. The bacteria contained a third glutamine synthetase, GSIII, which has been purified and partially characterized. GSIII had considerable glutamine synthetase activity when assayed using a semibiosynthetic (glutamate- and hydroxylamine-dependent) assay, but had no detectable transferase (glutamine- and hydroxyl-amine-dependent) activity. GSIII was inhibited by ADP and pyrophosphate but not by various nitrogen-containing metabolites that inhibit other GS enzymes. Activity was also inhibited by methionine sulfoximine, a transition state analog, but the concentration needed to inhibit GSIII was 50 to 100 times higher than that needed to inhibit GSI or GSII. GSIII had a Km for glutamate of 13.3 mM, for ammonium of 33 mM, and for hydroxylamine of 5.3 mM with a pH optimum of 6.8 and a temperature optimum of 50 degrees C. The purified protein had related subunits of 46.5 and 49 kDa and a native molecular mass of 355 kDa, indicating the native enzyme was an octamer. Polyclonal antibodies specific for GSIII reacted with a protein of similar molecular weight in Escherichia coli strains that carry R. meliloti glnT on a plasmid. GSIII activity was detected in some of these strains that contained glnT. Extracts of root nodules formed by WSU650 also react with the antibodies.
Shatters
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Department of Genetics and Cell Biology, Washington State University, Pullman 99164-6340.
Liu
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Kahn
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ML
D001217:Aspartate-Ammonia Ligase / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005800:Genes, Fungal; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005972:Glutaminase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics
D007527:Isoenzymes; D046911:Macromolecular Substances; D005972:Glutaminase; D001217:Aspartate-Ammonia Ligase; D005974:Glutamate-Ammonia Ligase