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Abstract

Fully unadenylylated glutamine synthetase (GS) from the endophytic bacterium Azospirillum brasilense Sp245 was isolated and purified. The enzyme was electrophoretically homogeneous and contained strongly bound metal ions, which could not be removed by dialysis. Mn2+, Mg2+, and Co2+ were found to be effective in supporting biosynthetic activity of the A. brasilense GS. Some kinetic properties of Mn2+-activated and Mg2+-activated unadenylylated GS were characterized. Circular dichroism analysis of the enzyme showed that the A. brasilense GS is a highly structured protein: 59% of its residues form alpha-helices and 13% beta-strands. Removal of the metal ions from the A. brasilense GS by treatment with EDTA resulted in alterations in the enzyme secondary structure.

Trace cobalt speciation in bacteria and at enzymic active sites using emission Mössbauer spectroscopy

“…Bacterial GSs are dodecameric proteins; the GS molecule is formed from two face-to-face hexameric rings of total 12 subunits with 12 active sites between the monomers, each having two divalent cation-binding sites, n1 and n2, which differ in their affinity for divalent cations [31]. For all GSs, including that of A. brasilense, divalent cations (commonly, Mg 2+ , Mn 2+ or Co 2+ ) are necessary for the activity to be expressed [9,20,30,31,32]. Along with the twelve n1 sites and twelve n2 sites, GS from E. coli has 48 additional metal-binding sites per oligomer with relatively lower affinity [32].…”

“…Azospirillum brasilense Sp245 was cultivated in a standard mineral medium as described elsewhere [9,18,19,20], with 5 mmol L -1 NH 4 Cl as a nitrogen source and 0.6% sodium malate as a carbon source (pH 6.9). For EMS studies of Co 2+ -treated cells, the bacterial culture was grown up to ca.…”

“…To isolate GS, A. brasilense Sp245 cells were disrupted by passing them through a French press [20]. They were then treated with 0.25 mg mL -1 DNAse I (Sigma) and 2 mg mL -1 streptomycin sulfate, centrifuged, and the supernatant was subjected, first, to ion-exchange chromatography in 50 mmol L -1 Tris-HCl buffer (pH 7.0) with a linear NaCl gradient from 0 to 1 mol L -1 using DEAE-Toyopearl 650 M (Toyo Soda), and then to FPLC on a Mono-Q column (Pharmacia Biotech) with the same buffer and NaCl gradient.…”

“…They were then treated with 0.25 mg mL -1 DNAse I (Sigma) and 2 mg mL -1 streptomycin sulfate, centrifuged, and the supernatant was subjected, first, to ion-exchange chromatography in 50 mmol L -1 Tris-HCl buffer (pH 7.0) with a linear NaCl gradient from 0 to 1 mol L -1 using DEAE-Toyopearl 650 M (Toyo Soda), and then to FPLC on a Mono-Q column (Pharmacia Biotech) with the same buffer and NaCl gradient. The homogeneous GS preparation obtained was treated for 30 min with 5 mmol L -1 EDTA (to remove metal cations, bound to native GS [20], from the enzyme) and then dialysed against 1000 volumes of 50 mmol L -1 KCl (pH 7.0) at 4°C to separate the enzyme from EDTA-metal chelates. Carrier-free 57 CoCl 2 (1 mCi) was added to 1 mL of aqueous solution of the resulting cation-free enzyme up to a ratio of ca.…”

“…Co 2+ was also shown to support the activity of glutamine synthetase (GS, a key enzyme of nitrogen metabolism) from this bacterium [9,20]. In the present work, Co 2+ metabolism in A. brasilense Sp245 was followed using EMS of bacterial cells (stored in aqueous suspensions with added traces of 57 CoCl 2 and frozen after certain periods of time).…”