Structural characterization of glutamine synthetase from Azospirillum brasilense

Abstract

CD spectroscopic study of the secondary structure of partly adenylylated glutamine synthetase (GS) of the bacterium Azospirillum brasilense showed both the native and cation-free (EDTA-treated) enzyme to be highly structured (58 and 49% as alpha-helices, 10 and 20% as beta-structure, respectively). Mg(2+), Mn(2+), or Co(2+), when added to the native GS, had little effect on its CD spectrum, whereas their effects on the cation-free GS were more pronounced. Emission ((57)Co) Mössbauer spectroscopic (EMS) study of (57)Co(2+)-doped cation-free GS in frozen solution and in the dried state gave similar spectra and Mössbauer parameters for the corresponding spectral components, reflecting the ability of the Co(2+)-enzyme complex to retain its properties upon drying. The EMS data show that (a) A. brasilense GS has 2 cation-binding sites per active center and (b) one site has a higher affinity to Co(2+) than the other, in line with the data on other bacterial GSs.

Application of 57Co emission Mössbauer spectroscopy to studying biocomplexes in frozen solutions

“…Preparation of Co 2+ -containing samples of glutamine synthetase (GS), a ubiquitous key enzyme of nitrogen metabolism [9] isolated from the nitrogen-fixing plant-associated rhizobacterium Azospirillum brasilense (strain Sp245), was described in detail elsewhere [10,11]. Treatment of GS with 5 mM EDTA for 30 min with further dialysis led to the removal of native cations from the enzyme (with subsequent loss of its activity in the absence of divalent cations in the medium).…”

“…Thus the coordination of Co and its distribution between the sites can be analysed. This is illustrated by EMS data on 57 Co 2+ -doped glutamine synthetase isolated from A. brasilense [10,11] ( Figure 2; see also Table I). …”

“…This is in good agreement with the existence of two separate cation-binding sites at each of the 12 active centres of the GS molecule, viz. a more symmetric site (with three Glu residues as ligands) and a less symmetric site with a significantly lower affinity to the cation (with one His and two Glu residues as ligands) [9][10][11]. In line with this conclusion, the former site (with a higher affinity for cations and Δ≈2.4 mm/s) may be expected to be primarily saturated with 57 Co 2+ (featured by a higher S r value), whereas the latter site (with Δ≈3.1 mm/s) binds the remaining 57 Co 2+ characterised by a lower S r value (see Table I).…”

“…The structural organisation of enzymic cation-binding sites (bearing Co 2+ alone or together with other cations, as in cases of double-site active centres related to two-metal-ion catalysis [16]) can also be probed by EMS using 57 Co 2+ -doped enzyme samples. Thus the coordination of Co and its distribution between the sites can be analysed.…”

“…Earlier we have shown that, along with Mg 2+ Figure 2 Emission Mössbauer spectrum of 57 Co 2+ -doped glutamine synthetase from A. brasilense in frozen aqueous solution (T=80 K). and Mn 2+ , cobalt(II) is one of the main activating cations for the A. brasilense GS, also playing a role in maintaining its secondary structure [10,11].…”