The physiological regulation of glutamine synthetase (GS; EC 6.3.1.2) in the axenic Prochlorococcus sp. strain PCC 9511 was studied. GS activity and antigen concentration were measured using the transferase and biosynthetic assays and the electroimmunoassay, respectively. GS activity decreased when cells were subjected to nitrogen starvation or cultured with oxidized nitrogen sources, which proved to be nonusable for Prochlorococcus growth. The GS activity in cultures subjected to long-term phosphorus starvation was lower than that in equivalent nitrogen-starved cultures. Azaserine, an inhibitor of glutamate synthase, provoked an increase in enzymatic activity, suggesting that glutamine is not involved in GS regulation. Darkness did not affect GS activity significantly, while the addition of diuron provoked GS inactivation. GS protein determination showed that azaserine induces an increase in the concentration of the enzyme. The unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of Prochlorococcus for coping with a light-and nutrient-limited environment.Prochlorococcus is a marine photosynthetic prokaryote ubiquitous in most intertropical areas of the oceans and is responsible for a significant part of the global primary production (for a review, see reference 27). Its unusual photosynthetic apparatus (12,13,15,29) and wide genetic diversity have attracted increasing scientific interest in recent years. Its ability to tolerate a very wide light gradient has been linked to the cooccurrence in the field of two ecotypes with distinct irradiance optima for growth and photosynthesis (21,40). While many reports have described these features in detail, little is known about other important aspects of Prochlorococcus metabolism, such as nutrient assimilation. In particular, the nitrogen assimilatory pathways in Prochlorococcus have not yet been studied. However, it is widely accepted that nitrogen is the main limiting nutrient in the upper layer of the oceans (27), and an understanding of the mechanisms involved in nitrogen assimilation could thus provide some keys to unveiling the remarkable ability of Prochlorococcus to colonize very oligotrophic regions. Nevertheless, such studies with Prochlorococcus face two problems: first, this organism is not easy to cultivate, and second, most isolated strains or clones contain contaminant heterotrophic bacteria. Only very recently, Rippka and coworkers described the first axenic strain, PCC 9511 (31), a typical high-light-adapted Prochlorococcus ecotype, allowing proper study of nonphotosynthetic metabolic pathways.In the present work, we have studied the physiological response of glutamine synthetase (GS) in cultures of Prochlorococcus subjected to different conditions by measuring transferase and biosynthetic activities and antigen concentration. The standard nitrogen assimilatory pathway in non-nitrogenfixing cyanobacteria is composed of a complex, highly modulated system of transporters, enzymes, and regulatory proteins (6). It allows...

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Limonin D-ring lactone hydrolase, the enzyme catalysing the reversible lactonization/hydrolysis of D-ring in limonin, has been purified from Citrus seeds and immobilized on Q-Sepharose to produce homogeneous limonoate A-ring lactone solutions. The immobilized limonin D-ring lactone hydrolase showed a good operational stability and was stable after sixty-seventy operations and storing at 4°C for six months.

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Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography. The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each). The isoelectric point is 4.9 as determined by isoelectric focusing. The homogeneous enzyme was used to determine the NH 2-terminal amino acid sequence. The enzyme was purified from cells grown in either fructose or limonoate as a carbon source. Limonoate dehydrogenase activity was higher in limonoate-grown cultures. Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD ؉. In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays. Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase. The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect.

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An open reading frame encoding a polypeptide of significant homology (55.7% identity) with the enoyl-CoA hydratase encoded by the gene echA1 from Mycobacterium tuberculosis has been found in the genome of the plant-pathogen bacteria Rhodococcus fascians strain NRRL-B-15096. Sequence alignments showed that it possesses several conserved blocks common to E. coli, M. tuberculosis and human mitochondria. One of such blocks includes a glutamate residue located at position 149, corresponding to the glutamate 139 of Escherichia coli. This glutamate was previously shown to be the catalytic residue of enoyl-CoA hydratase in the multienzyme complex of fatty acid oxidation from E. coli. Our results provide additional information on the conserved domains of this enzyme. Significant homologies in other genome regions between R. fascians and M. tuberculosis confirm their phylogenetic relationship.