Occurrence of Glutamine-2-Oxoacid Trans- Aminase Activity in the Blue-Green Alga Anabaena Cylindrica

J. Gen. App!. Microbiol., 31,171-176 (1985)

OCCURRENCE OF GLUTAMINE-2-OXOACID TRANS- AMINASE ACTIVITY IN THE BLUE-GREEN ALGA ANABAENA CYLINDRICA

MASAYUKI OHMORI, TOSHIRO SAINO, AND KAZUKO OHMORI*

Ocean Research Institute, University of Tokyo, Nakano-ku, Tokyo 164 *Faculty of Domestic Sciences , Showa Women's University, Setagaya-ku, Tokyo 155, Japan

(Received April 11, 1985)

Aspartate and glutamate were formed from glutamine and oxaloacetate with crude enzyme extracts obtained by ammonium sulfate precipitation (35-65 % saturation) from the blue-green alga Anabaena cylindrica. In this reaction the 2-amino group of glutamine was transferred to oxaloacetate in the absence of a reluctant. Azaserine, an inhibitor of glutamine-amide transfer, inhibited glutamate formation from glutamine but did not inhibit aspartate formation. Thus, a transamination from glutamine directly to oxaloacetate was indicated. This transamination reaction may be accomplished with glutamine-2-oxoacid transaminase which is found in animal tissues and in yeast.

Glutamine is a primary product in the assimilation of ammonia by blue-green algae and is further metabolized by glutamate synthase which transfers the amide nitrogen of glutamine to 2-oxoglutarate, forming two molecules of glutamate (1, 2). Glutamine can be degraded into glutamate and ammonia by glutaminase (L- glutamine amidohydrolase, EC 3.5.1.2), though the presence of this enzyme in blue-green algae has not been clearly demonstrated. In addition, a glutamine- 2-oxoacid transaminase (L-glutamine : 2-oxoacid aminotransferase, EC 2.6.1.15), which transfers the 2-amino group of glutamine directly to 2-oxoacid, has been reported in animals (3) and in yeast (4). This enzyme may also occur in bacteria (5). There has been no investigation of glutamine-2-oxoacid transaminase in blue- green algae. The results presented here suggest the occurrence of this enzyme together with glutaminase in Anabaena cylindrica.

MATERIALS AND METHODS

Anabaena cylindrica (strain Ml, from the culture collection of the Institute of

171 172 OHMORI, SAIN0, and OHMORI VOL. 31

Applied Microbiology, University of Tokyo) was grown under nitrogen-fixing conditions as previously reported (6). About 5 1 of four-day-old cells were harvested, washed twice with nitrogen-free fresh culture medium, and resus- pended in 60 ml of 100 mM Tris-HCJ buffer (pH 7.6) containing 1 mM EDTA and 6 mM mercaptoethanol. The cells were then sonicated with a Kubota-Insonator 200 M sonicator for a total of 10 min (10 times, l min each) and centrifuged for 20 min at 20,000 x g. Then the supernatant was subjected to ammonium sulfate precipitation. The protein precipitating between 35 and 65 % saturation of ammonium sulfate was dissolved in 20 mM Tris-HC1 buffer (pH 7.6), containing 1 mM EDTA and 6 mM mercaptoethanol, and dialyzed against 2 l of the same buffer over night. The dialysis was repeated, and the dialyzed sample was used as the crude enzyme. All procedures were carried out at 4°C. The activity of aspartate formation from glutamine and oxaloacetate was determined in a reaction mixture containing, unless otherwise stated, 0.5 mM glutamine, 5 mM oxaloacetate, 100 mM Tris-HC1 buffer (pH 7.6), 1 mM EDTA, 1 mM pyridoxalphosphate and 2 ml crude enzyme (about 20 mg protein) in a total volume of 3 ml. In the determination of glutamate formation from glutamine (glutaminase activity), oxaloacetate was omitted. The reaction was con- ducted at 25°C, and, at appropriate time intervals, 0.9-ml aliquots were removed and mixed with 0.3 ml of 20 % trichloroacetic acid (TCA) to stop the reaction. Then the samples were centrifuged and the supernatants were washed 5 times with ethyl ether to remove the TCA. The amino acids formed by the enzymic reaction were determined using a Hitachi 835 amino acid analyzer. The transfer of nitrogen from glutamine to aspartate was determined using an '5N-amide (50 atom %) labeled glutamine. After the enzymic reaction, amino acids were extracted as described above and then separated with two-dimensional glass-fiber paper chromatography as reported previously (7). The aspartate spot was cut out, dried and its 15N content was determined using an Anelva TE 360 quadrupole mass spectrometer (8). The transamination from glutamine to glyoxylate was determined according to the method of COOPERand MEISTER(9). The assay system contained in a final volume of 1.0 ml, 20 mM sodium glyoxylate, 20 mM uniformly labeled L-[14C]- glutamine (approximately 100,000 cpm), 50 mM Tris-HC1 buffer (pH 8.4) and 0.7 ml crude enzyme (about 8 mg protein). The reaction mixture was incubated at 25°C in a 5-ml Erlenmeyer flask and, at 20 min intervals, the reaction was stopped by adding 0.05 ml of 1 N H2S04. The mixture was then transferred quantitatively to the outside well of a Warburg flask, the inside well of which contained 0.05 ml of freshly prepared 5 N NaOH and a small piece of Wattman filter paper. After- wards, 0.1 ml of 2 N Ce(S04)2 was introduced into the outside well of the flask to evolve 14C02, and the flask was then closed and shaken slowly for 1 hr. Finally, the filter paper and NaOH solution with the absorbed 14C02 were transferred to a 10 ml liquid scintillation vial. The radioactivity of the sample was determined 1985 Glutamine Transaminase in Blue-green Algae 173 using a Packard Tri-Carb 3255 liquid scintillation counter.

RESULTSAND DISCUSSIONS When glutamine was used as an amino donor and oxaloacetate as an amino acceptor in the absence of a reluctant such as NADPH, aspartate and glutamate were formed linearly with time for at least 30 min. The activity of aspartate formation was hyperbolic with respect to glutamine concentration, and the apparent Km value for glutamine is about 3 x 10_4 M (Fig. 1). To determine whether aspartate-nitrogen was derived from the amide-N or the amino-N of glutamine, the 15Ncontent of the aspartate formed from 15N-amide (50 atom % 15N) glutamine and oxaloacetate was measured. If amide-N had been transferred to oxaloacetate, a very high 15N enrichment (almost 50 atom % 15N) in the formed aspartate would have been expected. However, it was found that the 15N content in the aspartate was very low (0.5 atom % i5N), suggesting that the amino-N of glutamine but not amide-N was the source of the aspartate-nitrogen. The direct transfer of a 2-amino group of glutamine to a 2-oxo group of an organic acid produces deaminated glutamine (2-oxoglutaramate) and amino acid, and one 2-oxoglutaramate releases one C02 by decarboxylation when exposed to ceric sulfate (10). To determine the formation of 2-oxoglutaramate from glutamine, 14C02 production in the presence of [U-14C]glutamine and glyoxylate was measured. Table 1 clearly shows that 2-oxoglutaramate was formed in this

Fig. 1. The effect of glutamine concentration on the rate of aspartate formation with the crude enzyme extracts of Anabaena cylindrica. Figure inset shows a Linewe- aver-Burk plot. Experimental conditions were as described in MATERIALS AND METHODS. 174 OHMORI, SAIN0, and OHMORI VOL. 31

Table 1. Formation of [14C]2-oxoglutaramate from L-[U-14C]glutamine with the crude enzyme extracts of Anabaena cylindrica.

reaction suggesting the presence of glutamine-2-oxoacid transaminase. The production of glycine from glutamine and glyoxylate was confirmed in separate ex- periments. There is still a possibility that aspartate was formed by transamination from glutamate derived from glutamine by glutaminase activity. Table 2 shows that the glutamate formation in the absence of 2-oxoacid (glutaminase activity) was dramatically inhibited by 1 mM azaserine, an inhibitor of amide transfer, but not by 1 mM aminooxyacetate, an inhibitor of transamination. By contrast, aspartate formation in the presence of oxaloacetate was completely inhibited by 1 mM aminooxyacetate but not by 1 mM azaserine (Table 3). Also 6-diazo-5-oxo-L-norleucine (DON), another inhibitor of glutaminase, did not inhibit aspartate formation (data not shown). These results suggest that direct transfer of the amino moiety of glutamine to oxaloacetate occurred in this reaction and that aspartate formation by glutaminase plus glutamate-aspartate transaminase was less significant. Table 4 shows that adding 2-oxoglutarate greatly stimulated glutamate formation. If glutaminase was the single enzyme responsible for glutamate production in our experimental conditions, no net increase in glutamate concentration would be expected. The reason is that the transamination from one glutamate molecule formed by glutaminase to one 2-oxoglutarate molecule results in the dis- 1985 Glutamine Transaminase in Blue-green Algae 175

Table 3. Effect of aminooxyacetate and azaserine on aspartate formation with the crude enzyme extracts of 4nabaena cylindrica.

appearance of one glutamate molecule and the production of one new glutamate molecule. Therefore, the strong enhancement of glutamate formation by 2-oxoglutarate supports the idea that direct amino transfer from glutamine to 2-oxoglutarate was carried out by a glutamine-2-oxoacid transaminase found in this protein fraction. In animal tissues the importance of glutamine transaminase in glutamine utilization has long been argued (3). In Neurospora crassa aerial mycelium with no glutaminase activity, glutamate and other amino acids were postulated to have been formed through the glutamine-2-oxoacid transaminase-w-aminase pathway (11). CRDENAS et al. (4) proposed a glutamine cycle in which glutamine transaminase, glutamate dehydrogenase and glutamine synthetase were included in the utilization and reformation of glutamine in the nitrogen-starved conidia of Neu- rospora crassa. In blue-green algae, glutamate synthase activity depends on reduced ferredoxin (12), and the reduction of ferredoxin depends on light. However, nitrogen assimilation proceeds during dark periods, and the accumulation of high glutamine concentrations have been observed in the presence of ammonia in the dark (13). This indicates that glutamate synthesis in the dark proceeds at a very slow rate com- pared with glutamine synthesis. Under these circumstances, it seems likely that glutamate and other amino acids are formed either by glutamine transaminase or by glutaminase plus glutamate-2-oxoacid transaminase, without using a reducing 176 OHMORI, SAINO, and OHMORI VoL. 31 power such as reduced ferredoxin.

We express our sincere thanks to Prof. A. Hattori of the Ocean Research Institute, University of Tokyo, for his stimulating discussions, Mr. J. Kanda for his help in 15N analysis and Dr. N. L. Collie for reviewing the manuscript. Part of this work was carried out at the Radioisotope Centre, University of Tokyo, and we are grateful to Dr. Y. Nakamura, of the Centre, for his in- valuable advise in the isotope work. This work was supported by a grant (No. 58390012) to M. 0. from the Ministry of Education, Science and Culture of Japan.

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