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Home , Aspartate dehydrogenase, Oxaloacetic acid

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 dehydrogenation, catalyzed by NADP+ -dependent aspartate dehydrogenase, in vitamin B12-producing Klebsiella pneumoniae IFO 13541. The , which was purified from a crude extract of K. pneumoniae IFO 13541, catalyzes the oxidative deamination of aspartic acid to form . This enzyme had a molecular mass of about 124kDa consisting of two identical subunits. L-Aspartic acid was a , although D-aspartic acid and L- 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 of aspartic acid in the vitamin B12 production (1-3). On the other hand, a variety of L- 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 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

. 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: (290kDa),

(142kDa), (67kDa), adenylate kinase (32kDa), and (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: , , glutamic acid, , , , , , , , , , , , , , and .

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 . 2). The optimum pH for the oxidative deamination of L-aspartic acid was about 7.0 in phosphate buffer. The optimum pH for the reaction rate in Tris-HCl buffer was

Table 1. Effect of various compounds on aspartate dehydrogenase .

The reaction mixture contained 40ƒÊL of 1M potassium phosphate buffer (pH 7 .0), 100ƒÊL of 0.1M amino acid, 50ƒÊL of 20ƒÊmol/mL NADP+ , and 0.35mg of crude extract (after dialysis) in a total volume of 600ƒÊL. FMN, FAD, and EDTA were added to 50ƒÊL at 20ƒÊmol/mL, 20ƒÊmol/mL, and 12ƒÊmol/mL, respectively .

J Nutr Sci Vitaminol Aspartate Dehydrogenase 487

Fig. 1. Stoichiometry of aspartate dehydrogenase. The reaction mixture contained

3.2mL of 250mM Tris-HCl buffer (pH 7.0), 2.0mL of 0.1M aspartic acid , 1.0mL of 12mM MgCl2, 1.0mL of 20mM NADP+, and 7mg of crude extract in a total

volume of 12mL. The reaction was stopped with 0.4mL of 2N HCl per 2mL of the

reaction mixture. •›, aspartic acid; •œ, oxaloacetic acid; • , ammonia .

Fig. 2. Effect of pH on aspartate dehydrogenase. •›, Tris-HCl buffer; • , potassium

phosphate buffer.

pH 8.0. The enzyme activity in Tris-HCl buffer was about 7-fold higher than that in phosphate buffer. It may be caused by inhibition of potassium ion in the phosphate buffer.

The thermostability of the enzyme was examined. When aspartate dehydro

genase was incubated at various temperatures for 10min, the enzyme retained its full activity up to 20•Ž, but it lost 70% of the activity at 50•Ž and the entire activity at 60•Ž (Fig. 3).

Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis of the native enzyme followed by activity staining of aspartate dehydrogenase is shown in Fig. 4. The electrophoresis of

purified aspartate dehydrogenase showed a single but broad band on activity staining.

Vol 44, No 4, 1998 488 T OKAMURA et al

Fig. 3. Effect of temperature on aspartate dehydrogenase stability. The reaction mix ture contained 40ƒÊL of Tris-HCI buffer (pH 7.5), 100ƒÊL of 0.1M aspartic acid, 50ƒÊL of 12mm MgCl2, 50ƒÊL of 20mm NADP+, and 0.35mg of crude extract in a total volume of 600ƒÊL.

Fig. 4. Activity staining. The activity staining of aspartate dehydrogenase was ex amined by the method described in Materials and Methods.

Molecular mass and subunit structure The molecular mass of the enzyme was estimated to be about 124kDa by gel filtration on a TSK gel G3000SW column (Fig. 5(A)). The molecular mass of the subunit was measured to be 62kDa by SDS-polyacrylamide gel electrophoresis (Fig. 5(B)). These results suggest that the enzyme is composed of two subunits identical in molecular mass.

Effects of anaerobic culture conditions on aspartate dehydrogenase production Figure 6 shows the relation of cell growth to production of aspartate de hydrogenase in the medium. More aspartate dehydrogenase was produced under anaerobic conditions than under aerobic conditions.

DISCUSSION

Until today, 12 kinds of amino acid dehydrogenase have been known: alanine dehydrogenase, glutamate dehydrogenase, serine dehydrogenase, valine dehydro genase, leucine dehydrogenase, glycine dehydrogenase, L-erythro-3, 5-diamino

J Nutr Sci Vitaminol Aspartate Dehydrogenase 489

Fig. 5. Molecular weight measurement of aspartate dehydrogenase by gel filtration on a TSK gel G3000SW column (A) and by SDS-PAGE (B). (A) and (B), the arrow indicates the position of aspartate dehydrogenase.

Fig. 6. Time course of production of aspartate dehydrogenase by K. pneumoniae IFO

13541. K. pneumoniae was incubated at 30•Ž in the medium under aerobic and

anaerobic conditions. The aspartate dehydrogenase production of the cells was

examined by the method described in Materials and Methods. Growth was under

aerobic (•›) and anaerobic (•œ) conditions; specific activity of aspartate

dehydrogenase was under aerobic (_??_) and anaerobic (_??_) conditions. hexanoate dehydrogenase, 2,4-diaminopentanoate dehydrogenase, lysine dehydro genase, meso-2,6-diaminopimelate dehydrogenase, phenylalanine dehydrogenase, and tryptophan dehydrogenase (4). However, there had been no information on aspartate dehydrogenase. In this study, we found a new reaction of aspartic acid dehydrogenation by

Vol 44, No 4, 1998 490 T OKAMURA et al

NADP+ -dependent aspartate dehydrogenase. The enzyme catalyzes the oxidative deamination of aspartic acid to form oxaloacetic acid, in vitamin B12-producing K. pneumoniae IFO 13541.

REFERENCES

1) Ohsugi M, Yamada M, Yoshida Y, Ishibashi F, Inoue Y. 1983. Isolation of a formate assimilating bacterium and its vitamin B12 formation. Agric Biol Chem 47: 1127-1128. 2) Ohsugi M, Noda H, Muro K, Ishiba A, Kondo Y, Nakao S. 1989. Effects of the yeast extract components pyrroloquinoline quinone and aspartic acid on vitamin B12 production in Klebsiella pneumoniae IFO 13541. J Nutr Sci Vitaminol 35: 661-665. 3) Ohsugi M, Noda H, Nakao S. 1993. Participation of aspartic acid and pyrroloquinoline quinone in vitamin B12 production in Klebsiella pneumoniae IFO 13541. J Nutr Sci Vitaminol 39: 323-333. 4) Ohshima T, Soda, K. 1990. Biochemistry and biotechnology of amino acid dehydrogenase. Adv Biochem Eng Biotechnol 42: 187-208. 5) Hummel W, Kula RM. 1989. Dehydrogenases for the synthesis of chiral compounds. Ear J Biochem 184: 1-13. 6) Ohshima T, Yamazaki T. 1990. Dye-ligand affinity chromatography-Simple preparation method of dye ligand resins and their application. Seikagaku 62: 283-287. 7) Lowry HO, Rosebrough JN, Farr LA, Randall JR. 1951. Protein measurement with the folio phenol reagent. J Biol Chem 193: 265-275. 8) Davis JB. 1964. Disc electrophoresis-II; Method and application to human serum proteins. Ann NY Acad Sci 121: 404-427. 9) Weber K, Osborn M. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244: 4404-4412. 10) Brady GT. 1961. Conway microdiffusion technique. In: Biochemists' Handbook (Long C, ed), p 165-172. E & FN Spon, London.

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