Site-directed mutagenesis of Glu-297 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase alters kinetic and structural properties and confers resistance to L-methionine sulfoximine.

In this paper we examine the functionality of Glu-297 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase (EC 6.3.1.2). For this purpose, the gln alpha cDNA was recombinantly expressed in Escherichia coli, and site-directed mutants constructed, in which this residue was replaced by alanine. The level of glutamine synthetase transferase catalytic activity in the mutant strain was 70-fold lower while biosynthetic activity remained practically unaffected. Kinetic parameters for both enzyme activities were not greatly altered except for the Km for ammonium in biosynthetic activity, which increased 100-fold. A similar result was reported when mutagenizing Glu-327 from E. coli glutamine synthetase, a residue shown to be present at the active site. This suggests that the Glu residue mutated in the higher-plant enzyme could develop a similar catalytic role to that of bacteria. Another characteristic feature of the mutant protein was its higher resistance to inhibition of the biosynthetic activity by L-methionine sulfoximine, a typical inhibitor of glutamine synthetase. In addition, we show that immunoreactivity of the glutamine synthetase mutant protein, both under native and denaturing conditions, is similar to the wild type, indicating that no deep conformational changes were produced as a consequence of the introduced mutation. However, structural changes in the active site can be predicted from alterations detected in the behaviour of the mutant protein towards affinity chromatography on 2',5'-ADP-Sepharose, as compared to the wild type. Nevertheless, complementation of an E. coli glnA mutation indicated that the E297A mutant enzyme was physiologically functional.

Functional importance of Asp56 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase. An essential residue for transferase but not for biosynthetic enzyme activity.

Replacement of Asp56 by site-directed mutagenesis of the alpha-gene from Phaseolus vulgaris glutamine synthetase heterologously expressed in Escherichia coli produces a complete loss of transferase enzyme activity, thus revealing essentiality of the residue for this particular enzyme activity. This happens independent of Asp56 being replaced by Ala or Glu, suggesting that the essentiality of this residue cannot be attributed to its negative electrical charge. However, a high level of glutamine synthetase biosynthetic specific activity (referred to glutamine synthetase protein, as determined immunologically), is present in D56A and D56E mutants, suggesting that Asp56 is an example of a residue that has a different role in the catalytic mechanism of both enzyme activities of this protein. Km for ATP, glutamate and Mg2+, as well as energy of activation, can be altered as a consequence of the performed mutations. However, the Km and catalytic efficiency for ammonium remains unaffected. Therefore, the catalytic role of Asp56 in the alpha-polypeptide of higher plant glutamine synthetase is quite different from the role proposed for its highly conserved homologue in bacteria (Asp50 in E. coli), which has been associated with binding and deprotonation of ammonium. On the other hand, we also show other results indicating that Asp56 is important in the spatial conformation of the active site and/or the protein, Asp56 being a crucial residue in the salting-out aggregation properties of the enzyme.

Regulation of the expression of ferredoxin-nitrite reductase in synchronous cultures of Chlamydomonas reinhardtii.

The regulation of ferredoxin-nitrite reductase--the second enzyme involved in the nitrate assimilatory pathway--in synchronous cultures of C. reinhardtii has been studied both at the activity and protein levels using specific antibodies. During a cycle of 12 h light/12 h dark (12L:12D), ferredoxin-nitrite reductase activity shows a 24-h fluctuation with a maximum in the middle of the light period. The increase of activity during the first few hours of the light phase is due to de novo synthesis of the enzyme. This synthesis occurs in the absence of NH4+ and it is highly induced by either nitrate or nitrite, but it does not require light so long as carbon skeletons are available. The decrease of ferredoxin-nitrite reductase activity during the last hours of the light period and during the dark phase is suggested to be due to protein degradation, although this process is slow because of the high stability of the enzyme. The changes in the level of ferredoxin-nitrite reductase seem to be related to events in the cell cycle under the illumination conditions used. Thus, synthesis of the enzyme correlates to growth periods within the cell cycle, and it does not seem to be under the control of a circadian rhythm.