Abstract
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.
Highlights
ADP and pyrophosphate but not by various nitrogencontaining metabolites that inhibit other Glutamine synthetase (GS) enzymes
We report a new glutamine synthetase activity incrude extracts of R. meliloti WSU650 that had both biosynthetic and semibiosynthetic activity but no detectable transferase activity
This paper presents the purification and partial characterization of GSIII from R. meliloti WSU650 and argues that this enzymatic activityallows WSU650 to grow without exogenous glutamine
Summary
(Sigma) to assay ammonia production. y-Glutamylphosphate reductase (GP-reductase) activity was assayed using the coupled assay of Extracts of a glnA glnII mutant of R. meliloti 104A14 Have. Up to 4 pg of purified protein were used in both Glutamine Synthetic and Semibiosynthetic Activity-Previous the glutaminase and theGP-reductase assays. Protein Purification-GSIII was purified from WSU650cells grown a t 30 "Cin 40 liters of minimal mannitol medium supplemented with 0.1% glutamate and 0.05% ammonium chloride. 100 g of bacteria were resuspended in 60 ml of IMG buffer (20 mM grows on minimal glutamate and ammonium medium at a rate of about one-half that of wild type R. meliloti 104A14 (Somerville et al, 1989). The pellet was resuspended in IMG buffer, dialyzed against gible transferase activity. Experiments with a defined minimal media showed that semibiosynthetic activity decreased about 5-fold when high concentrations of glutamine were present (data not shown). Purification and Analysis of GSIZI-GSIII, the enzyme responsible for the semibiosynthetic activity, was purified to homogeneity from R.
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