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Summary We Found a New Reaction of Aspartic Acid Dehydrogenation, Catalyzed by NADP+

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Summary We Found a New Reaction of Aspartic Acid Dehydrogenation, Catalyzed by NADP+ J Nutr Sci Vitaminol, 1998, 44, 483-490 Aspartate Dehydrogenase in Vitamin B12-Producing Klebsiella pneumoniae IFO 13541 Tokumitsu OKAMURA, Hiroko NODA, Shoko FUKUDA and Masahiro OHSUGI Department of Food Science and Nutrition, School of Human Environmental Sciences, Mukogawa Women's University, Nishinomiya 663-8137, Japan (Received February 2, 1998) Summary We found a new reaction of aspartic acid dehydrogenation, catalyzed by NADP+ -dependent aspartate dehydrogenase, in vitamin B12-producing Klebsiella pneumoniae IFO 13541. The enzyme, which was purified from a crude extract of K. pneumoniae IFO 13541, catalyzes the oxidative deamination of aspartic acid to form oxaloacetic acid. This enzyme had a molecular mass of about 124kDa consisting of two identical subunits. L-Aspartic acid was a substrate, although D-aspartic acid and L-glutamic acid were inactive. The enzyme showed maximal activity at about pH 7.0-8.0 for the oxidative deamination of L-aspartic acid, and it required NADP+ as a coenzyme, while NAD+ was inactive. Key Words aspartic acid, dehydrogenase, vitamin B12, Klebsiella pneumoniae We have previously reported on vitamin B12 production by Klebsiella pneumoniae IFO 13541, and we pointed out that the growth of the organism and the amount of vitamin B12 produced depended exclusively on the concentration of yeast extract added to the medium. The yeast extract components required were identified as aspartic acid and pyrroloquinoline quinone (PQQ). We have also reported the effects of aspartic acid and PQQ on the production of vitamin B12 by K pneumoniae IFO 13541 and the metabolism of aspartic acid in the vitamin B12 production (1-3). On the other hand, a variety of L-amino acid dehydrogenases have been extensively studied, as reviewed by Ohshima and Soda (4), and used for the synthesis or measurement of L-amino acids (4,5). However, the specific enzyme for L-aspartic acid dehydrogenation has not yet been identified. We found a new reaction of aspartic acid dehydrogenation, a novel NADP+ -dependent aspartate dehydrogenase, in crude extract of vitamin B12- producing K. pneumoniae IFO 13541. We describe here a characterization of the novel NADP+ dependent aspartate 483 484 T OKAMURA et al dehydrogenase from K. pneumoniae IFO 13541. MATERIALS AND METHODS Materials. NAD+, NADP+, NADH, and NADPH were obtained from Oriental Yeast; L-aspartic acid, D-aspartic acid, and L-glutamic acid were purchased from Nacalai Tesque. Other chemicals used were analytical grade reagents . Microorganism. The Klebsiella pneumoniae IFO 13541 used in this study was obtained from the Institute for Fermentation Osaka (IFO). Cultivation of K pneumoniae. The culture medium for K. pneumoniae con sisted of 0.5% meat extract, 1.0% peptone, 0.5% NaCl, and 0 .5% aspartic acid (pH 7.0). K. pneumoniae culture grown on slant was inoculated into 500mL of the medium in a 1L Erlenmyer flask. The cultivation was carried out at 30•Ž for 1d under aerobic conditions, or at 30•Ž for 4d under anaerobic conditions. Cells were collected by centrifugation at 10,000•~g for 30min and washed twice with an ice-cold saline solution. The cell pellet, suspended in 10mM potassium phosphate buffer (pH 7.0) containing 0.01% ƒÀ-mercaptoethanol, was subjected to sonication with an ultrasonic oscillator (BRANSON, 20kHz) for 16min at below 8•Ž. The undestroyed cells and debris were removed by centrifugation at 10 ,000•~g for 20min. The supernatant solution obtained was used as the cell-free extract. Pur fication of aspart ate dehydrogenase. All operations were performed at 0-10•Ž. As a standard buffer for purification, 10mM potassium phosphate pH 7.0 and pH 7.4 containing 0.01% ƒÀ-mercaptoethanol was used. Step 1. Preparation of crude extract: Cells (about 6g wet mass) were suspended in 150mL buffer (pH 7.0) and disrupted by sonication (12 times in 1min). Un broken cells and cell debris were removed by centrifugation (10,000•~g for 20min). The supernatant solution was used as the crude extract. Step 2. Protamine sulfate treatment: To the crude extract (145mL, 725mg of protein), 3.5mL of 1.5% protamine sulfate was added and stirred for 20min. The precipitate thus obtained was removed by centrifugation (10,000•~g for 20min). Step 3. Ammonium sulfate fractionation: The supernatant solution (140mL) was brought to 50% saturation with ammonium sulfate and the precipitate removed by centrifugation. Ammonium sulfate (80% saturation) was added to the super natant solution. The precipitate obtained by centrifugation was dissolved in a minimum volume of the pH 7.0 buffer and dialyzed against 20 volume of the same buffer. Step 4. DEAF-cellulose column chromatography: The enzyme solution (10mL, 69mg of protein, 50-80% ammonium sulfate fractionation) was applied to DEAE-cellulose column (0.8•~15cm) equilibrated with the pH 7.0 buffer. After the column was washed with pH 7.0 buffer supplemented with 0.1M NaCl , the retained enzyme was eluted with the same buffer supplemented with 0.2M NaCl. Step 5. Dye-ligand affinity chromatography: The enzyme solution (5mL , 20mg of protein) was applied to dye-ligand affinity column (6) (green-sepharose 4B, J Nutr Sci Vitaminol Aspartate Dehydrogenase 485 1.0•~2cm) equilibrated with the pH 7.4 buffer. After the column was washed with pH 7.4 buffer, the retained enzyme was eluted with the same buffer containing 2N NaCl. Step 6. High pressure liquid chromatography: The enzyme solution (0.5mL) was put on a TSK gel G3000SW column (0.75•~30cm) equilibrated with 0.1M potassium phosphate buffer (pH 7.4) containing 0.2M KCl, 10% glycerol, and 0.01% ƒÀ-mercaptoethanol. The column was equipped with a Tosoh HPLC system and developed at room temperature at a flow rate of 0.7mL/min with the same buffer. The active fractions were pooled and stored at -20•Ž in the presence of 40% glycerol. The enzyme was purified about 500-fold with 1% yield by these procedures . Enzyme assay. The standard reaction mixture contained 200ƒÊmol of L - aspartic acid (pH 7.0), 1ƒÊmol of NADP+, 200ƒÊmol of Tris-HCl (pH 7.0), and enzyme in a final volume of 1.0mL. The substrate was replaced by water in a blank. Incubation was done at 30•Ž in a cuvette with a 1-cm light path. The reaction was started by the addition of NADP+ and monitored by measuring the initial change in the absorbance at 340nm with a Hitachi 150-20 double beam spectrophotometer . One unit of the enzyme was defined as the amount that catalyzed the formation of 1ƒÊmol of NADPH per min in the reaction. Specific activity was expressed as units per mg of protein. Protein was measured by the method of Lowry et al. (7) with crystalline bovine serum albumin as the standard. Electrophoresis. Gel electrophoresis of the native enzyme was done with 7.5% polyacrylamide gel by the method of Davis (8). The enzymatic activity staining mixture contained 50mM Tris-HCl buffer (pH 7.5), 1.25mM NAD+, 10mM aspartic acid, 0.4mM phenazine methosulfate, and 0.5mM nitro blue tetrazolium. Protein was stained with 0.04% Coomassie brilliant blue R-250 in 3.5% perchloric acid. SDS-polyacrylamide gel electrophoresis was done with 10% polyacrylamide by the method of Weber and Osborn (9). Measurement of concentrations of aspartic acid, oxaloacetic acid, and ammonia. The reaction mixture contained 3.2mL of 250mM Tris-HC1 (pH 7.0), 2.0mL of 100mM aspartic acid, 1.0mL of 12mM MgCl2, 1.0mL of 20mM NADP+, and crude extract containing 7mg of protein in a total volume of 12mL. The reaction was stopped with 0.4mL of 2N HCl per 2mL of the reaction mixture. Aspartic acid, oxaloacetic acid, and ammonia were measured by the ninhydrin method (P. P. C.), hydrazine method, and Conway microdiffusion method, re spectively (10). Measurement of molecular mass. The molecular mass was estimated by gel fi ltration on a TSK gel G3000SW column (0.75•~30cm) at a flow rate of 0.7mL/min, with 0.1M potassium phosphate buffer (pH 7.4) containing 0.2M KCl, 0.01% ƒÀ-mercaptoethanol, and 10% glycerol . A calibration curve was made with the following proteins: glutamate dehydrogenase (290kDa), lactate dehydrogenase (142kDa), enolase (67kDa), adenylate kinase (32kDa), and cytochrome c (12.4 kDa). The molecular mass of the subunit was estimated by SDS-polyacrylamide Vol 44, No 4, 1998 486 T OKAMURA et al gel electrophoresis (8) with the following standard protein: MBP-ƒÀ-galactosidase (175kDa), MBP-paramyosin (83kDa), glutamate dehydrogenase (62kDa), ƒÀ- lactoglobulin A (25kDa), lysozyme (16.5 kDa), and aprotinin (6 .5 kDa). RESULTS Effect of various compounds on aspartic acid dehydrogenation As shown in Table 1, aspartic acid dehydrogenation was inactivated by boiling . NADP+ was the coenzyme for the oxidative deamination . Flavin coenzymes such as FMN and FAD did not show appreciable influence on the enzyme activity . The enzyme acted on L-aspartic acid in the oxidative deamination , and D-aspartic acid was inactive. The following amino acids (L-form) could not serve as substrates: glycine, alanine, glutamic acid, threonine, serine, leucine, isoleucine, methionine, cysteine, proline, valine, phenylalanine, tyrosine, tryptophan, lysine, histidine , and arginine. Stoichiometry of the reaction of aspartic acid dehydrogenation The Stoichiometry of the reaction for oxidative deamination is shown in Fig . 1. Aspartic acid was deaminated to form equimolar concentration of oxaloacetic acid and ammonia. The absorption spectrum of 2,4-dinitrophenylhydrazone of the oxaloacetic acid formed was identical with that of authentic oxaloacetic acid . Because the enzyme catalyzed the oxidative deamination of aspartic acid to form oxaloacetic acid, the enzyme was named aspartate dehydrogenase . Effects of pH and temperature on the enzyme activity The enzyme activity was determined in buffers of different pHs (Fig .
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