Characterization of glutamine synthetase from the marine diatom Skeletonema costatum

Glutamine synthetase (GS) plays a central role in nitrogen metabolism in photosynthetic eukuyotes. Two peaks of GS activity were resolved by anion-exchange chromatogmarinefrom the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity and this isoenzyme was purified over 200-fold. Results from denaturing gel electrophoresis and gel filtration chromatography suggest that six, 70-kD subunits comprise the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to a type of bacterial GS than to the type common among other eukaryotes. Western blot analysis of the two peak fractions from the anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later-eluting peak of enzyme activity. This form of GS does not appear unique to S. costatum as the antiserum recognized a similar sized protein in cell lysates of other chromophyte algae.; A cDNA encoding GS was isolated by PCR amplification. The predicted size of the encoded protein (45 kD) was smaller than the GS polypeptide (70 kD) identified by biochemical purification. Nucleic acid and deduced amino acid sequences of the diatom GS were greater than 50% identical to GS from green algae and vascular plants. The presence of a N-terminal signal sequence, identified based on sequence similarity with other chloroplast-localized proteins from diatoms, suggests that the encoded GS isoenzyme is localized to the chloroplasts. Phylogenetic analyses indicate that genes encoding GS from the diatom and two species of green algae diverged prior to the gene duplication giving rise to the isoenzymes in vascular plants and support the hypothesis that GS isoenzymes in diatoms, green algae and vascular plants arose through independent evolutionary event.; GS activity and isoenzyme abundance were examined during a transition in nitrogen supply. GS expression was examined using antisera raised against GS from S. costatum and two heterospecific GS antisera. Expression of the putative GS polypeptides varied in response to the nitrogen treatment. Further analysis of factors regulating the expression of the GS isoenzymes should provide insight into the physiological function of the isoenzymes.