Glutaminaa sintetasa

JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4732-4735 0021-9193/90/084732-04$02.00/0 Copyright C 1990, American Society for Microbiology

Vol. 172, No. 8

Purification and Properties of Glutamine Synthetases from the Cyanobacteria Synechocystis sp. Strain PCC 6803 and Calothrix sp. Strain PCC 7601
ANGEL MERIDA,1 LINE LEURENTOP,2 PEDRO CANDAU,' AND FRANCISCO J. FLORENCIOl* Departamento deBioquimica Vegetal y Biologia Molecular, Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla-CSIC, Apartado 1113, 41080 Seville, Spain,' and Department ofBiology, University ofAntwerp, B-2610 Wilrijk, Antwerp, Belgium2
Received 16 January 1990/Accepted 4 May 1990

Glutanine synthetases (GSs) from two cyanobacteria, one uniellular (Synechocystis sp. strain PCC 6803) and the otherflamentous (Calothrir sp. strain PCC 7601 [FremyeUla diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, icti that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an amonum-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted byammonium that had not been demonstrated previously in cyanobacteria.

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Ammonium assimilation takes place in cyanobacteria mainly by the sequential action of glutamine synthetase (GS) (L-glutamate:ammonia ligase [ADP-forming], EC 6.3.1.2) and glutamate synthase (L-glutamate:ferredoxin oxidoreductase [transaminating], EC1.4.7.1) (11). GSs purified from a variety of procaryotic sources are quite similar in physicochemical parameters; for example, they have a molecular mass of about 600 kilodaltons and have a similar number of subunits and similar structures (dodecamers, with a single subunit type of about 50 kilodaltons). By contrast, their regulatory properties differ widely. The enzymes of Escherichia coli (22) andother gram-negative bacteria (20) are regulated at the activity level by a covalent modification of the enzyme through an adenylylation-deadenylylation system. Most gram-positive bacteria lack this system and exhibit a feedback regulation by products such as glutamine and other metabolites (3). The regulation of cyanobacterial GS activity is not clear, although amino acids (acting in a cumulativemanner), divalent cations, and thiols have been proposed as modulators of the activity (6, 14, 19). There is no evidence of covalent modification of a GS from any cyanobacteria (4). GSs from cyanobacteria of the genera Anabaena (13, 17), Synechococcus (5), and, more recently, Phormidium have been purified to homogeneity (2, 18). All of them were similar in size and subunit composition. We havepurified GSs from the unicellular, facultative, heterotrophic cyanobacterium Synechocystis sp. strain PCC 6803 (section I) (15) and from the filamentous Calothrix sp. strain PCC 7601 (Fremyella diplosiphon) (section IV) (15) by a previously described method (5) that includes ion-exchange chromatography followed by affinity chromatography on 2',5'-ADPSepharose. In both cases, this procedure provides ahigh yield of enzyme (about 40%) that is pure by electrophoretic criteria; however, GS activity is clearly more abundant in the Calothrix sp. than in the Synechocystis sp. (2.45 versus 0.056 U/mg of protein in the crude extracts). We have carried out a structural study of Calothrix GS by using electron microscopy techniques and computerized image processing (9). This enzyme shows a typicalprocary*

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otic configuration, with a diameter of about 15 nm, and is formed by two superimposed hexagons, each composed of six subunits arranged radially with respect to the central hole but with a left-handed configuration. Rotational studies indicated that only rotation by 600 (or its multiples) gave a clear reinforcement of the intensities of the subunits (Fig. 1). In side...